Tissue Engineering and Transplantation in the Fetus

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Tissue Engineering and Transplantation in the Fetus Dario O. Fauza Associate Professor, Department of Surgery, Harvard Medical School and Boston Children’s Hospital, Boston, Massachusetts

INTRODUCTION The fetus is, arguably, the ideal tissue-engineering subject, both as a host and as a donor. The developmental and long-term impacts of tissue implantations into a fetus, along with the many unique characteristics of fetal cells, add new dimensions which greatly expand the reach of tissue engineering, to extents unmatched by most other age groups. Indeed, perhaps not surprisingly, attempts at harnessing these prospective benefits started long before the modern eras of transplantation and regenerative medicine. The first reported transplantation of human fetal tissue took place almost a century ago, in 1922, when a fetal adrenal graft was transplanted into a patient with Addison’s disease [1]. A few years later, in 1928, fetal pancreatic cells were transplanted in an effort to treat Diabetes mellitus [2]. In 1957, a fetal bone marrow transplantation program was first undertaken [3]. All those initial experiments involving human fetal tissue transplantation failed. It was only since the late twentieth century that fetal tissue transplantation in humans has started to yield favorable outcomes. A number of therapeutic applications of fetal tissue have already been explored, with variable results. Although the majority of studies to date have involved simply fetal cell, tissue, or organ transplantation, various engineered open systems using fetal cells have been explored in animal models and their first clinical applications are justifiably expected in the not too distant future. Fetal tissue has also been utilized as a valuable investigational tool in biomedical science, since the 1930s. Embryologists, anatomists and physiologists have long studied fetal metabolism, feto-placental unit function, premature life support and brain activity in pre-viable fetuses [4,5]. In vitro applications of fetal tissue are well established and somewhat common. Cultures of different fetal cell lines, as well as commercial preparations of human fetal tissue, have been routinely used in the study of normal human development and neoplasias, in genetic diagnosis, in viral isolation and culture and to produce vaccines. Biotechnology, pharmaceutical and cosmetic companies have employed fetal cells and extra-embryonic structures such as placenta, amnion and the umbilical cord to develop new products and to screen them for toxicity, teratogenicity and carcinogenicity. Fetal tissue banks have been operating in the United States and abroad for many years as source of various fetal cell lines for research. Considering that a large body of data has come out of research involving fetal cells or tissues, and that attempts at engineering virtually every mammalian tissue have already taken place, comparatively less has been done on the ’true’ engineering of fetal tissue, through culture and Principles of Tissue Engineering. http://dx.doi.org/10.1016/B978-0-12-398358-9.00026-4 Copyright Ó 2014 Elsevier Inc. All rights reserved.

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PART 5 Transplantation of Engineered Cells and Tissues placement of fetal cells into matrices or membranes, or through other in vitro manipulations prior to implantation. Controlled human trials of open system tissue engineering have yet to be performed and a relatively small number of animal experiments have been reported thus far. Fetal cells were first used experimentally in engineered constructs by Vacanti et al. [6]. Interestingly, this investigation was part of the introductory study on selective cell transplantation using bioabsorbable, synthetic polymers as matrices. This same group performed another study involving fetal cells in 1995 [7]. Both experiments, in rodents, did not include structural replacement, or functional studies. The use of fetal constructs as a means of structural and functional replacement in large animal models was first reported experimentally only in 1997 [8,9]. This chapter offers an outlook of the infantile field of fetal tissue engineering, along with a general overview of fetal cell and tissue transplantation.

GENERAL CHARACTERISTICS OF FETAL CELLS Immunological rejection (in non-autologous applications), growth limitations, differentiation and function restraints, incorporation barriers and cell/tissue delivery difficulties are all well known complications of tissue engineering. Many of those problems can be better managed, if not totally prevented, when fetal cells are used. Due to their properties both in vitro and in vivo, fetal cells are excellent building blocks for tissue engineering.

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Compared with cells harvested postnatally, most fetal cells multiply more rapidly and more often in culture. Depending on the cell line considered, however, this increased proliferation is more or less pronounced, or, in a few cases, not evident at all. Due, at least in part, to their proliferation and differentiation capacities, fetal cells have long been recognized as ideal targets for gene transfers. Because they are very plastic in their differentiation potential, fetal cells respond better than mature cells to environmental cues. Data from fetal myoblasts and osteoblasts, and from amniotic mesenchymal stem cells (aMSCs) suggest that purposeful manipulations in culture or in a bioreactor can be designed to steer fetal cells to produce improved constructs. Younger mesenchymal stem cells (MSCs) from mid-gestational fetal tissues are more plastic and grow faster than adult, bone marrow-derived MSCs. MSCs have also been isolated earlier in fetal development, from first-trimester blood, liver, and bone marrow. These cells are biologically closer to embryonic stem cells and have unique markers and characteristics not found in adult bone marrow MSCs, which are potentially advantageous for cell therapy. Fetal MSCs may express Human Leukocyte Antigen (HLA) class I but not HLA class II. The presence of interferon gamma in the growth medium may initiate the intracellular synthesis and cell surface expression of HLA class II, but neither undifferentiated nor differentiated fetal MSCs tend to induce proliferation of allogenic lymphocytes in mixed cultures. Actually, fetal MSCs treated with interferon-gamma typically suppress alloreactive lymphocytes in this setting. These and other data indicate that both undifferentiated and differentiated fetal MSCs may not elicit much alloreactive lymphocyte proliferation, thus potentially rendering these cells particularly suitable for heterologous transplantation. Fetal cells can survive at lower oxygen tensions than those tolerated by mature cells and are therefore more resistant to ischemia during in vitro manipulations. They also commonly lack long extensions and strong intercellular adhesions. Probably because of those characteristics, fetal cells display better survival after refrigeration and cryopreservation protocols when compared with adult cells. This enhanced endurance during cryopreservation, however, seems to be tissue specific. For instance, data from primates and humans have shown that fetal hematopoietic stem cells, as well as fetal lung, kidney, intestine, thyroid and brain tissues can

CHAPTER 26 Tissue Engineering and Transplantation in the Fetus

be well preserved at low temperatures, whereas non-hematopoietic liver and spleen tissues can also be cryopreserved, but not as easily.

In vivo The expression of major histocompatibility complex (H-2) antigens in the fetus and, hence, fetal allograft survival in immunocompetent recipients is age and tissue specific. The same applies to fetal allograft growth, maturation, and function. At least in fetal mice, the precise gestational time of detection of H-2 antigen expression and the proportion of cells expressing these determinants depend on inbred strain, specific haplotype, tissue of origin and antiserum batch employed. Nevertheless, the precise factors governing the timing and tissue-specificity of H-2 antigen expression are yet to be determined in most species, including humans. Other mechanisms, in addition to H-2 antigen expression, also seem to govern fetal immunogenicity. For example, at least in certain mammalian species, by catabolizing tryptophan the conceptus suppresses T cell activity and defends itself against rejection by the mother. In humans, fetal cells can be found in the maternal circulation in most pregnancies, and fetal progenitor cells have been found to persist in the circulation of women decades after child birth [10,11]. Interestingly, a certain population of fetal cells, the so-called pregnancy-associated progenitor cells (PAPCs), appears to differentiate in diseased/injured maternal tissue. The precise original phenotypical identity of these cells remains unknown. They are thought to be a hematopoietic stem cell, a MSC, or possibly a novel cell type. What is known is that pregnancy results in the acquisition of cells with stem-cell-like properties that may influence maternal health post-partum, by triggering disease and/or avoiding/combating it. All these data allow us to suppose that engineered constructs made with fetal cells should be less susceptible to rejection in allologous applications. Xenograft implantations may also become viable, as studies suggest that fetal cells are also better tolerated in cross-species transplantations, including in humans [12e14]. On the other hand, while less immunogenic, some fetal cells may be too immature and functionally limited, if harvested too early. Yet, experimental models of fetal islet pancreatic cell transplantation have shown that, with time, the initially immature and functionally limited cells will grow, develop and eventually function normally. Conversely, however, certain cells, such as those from the rat’s striatum, actually function better after implantation if harvested early, as opposed to late in gestation. Fetal cells may produce high levels of angiogenic and trophic factors, which enhance their ability to grow once grafted. By the same token, those factors may also facilitate regeneration of surrounding host tissues. Interestingly, significant clinical and hematological improvement has been described following fetal liver stem cell transplantation in humans, even when there is no evidence of engraftment. These improvements have been attributed to regeneration of autologous hematopoiesis and inhibition of tumor cell growth promoted by the infused cells, through mechanisms yet to be determined. The under-differentiated state of fetal cells also optimize engraftment, by allowing them to grow, elongate, migrate and establish functional connections with other cells [13].

Applications Because of all the general benefits derived from the use of fetal cells, along with others specific to each cell line, several types of fetal cellular transplantation have been investigated experimentally or employed in humans for decades now. Clinically, fetal cells have been (mostly anecdotally) useful in a number of different conditions, including: Parkinson’s and Huntington’s disease; Diabetes mellitus; aplastic anemia; Wiskott-Aldrich syndrome; thymic aplasia (DiGeorge syndrome) and thymic hypoplasia with abnormal immunoglobulin syndrome (Nezelof syndrome); thalassemia; Fanconi anemia; acute myelogenous and

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lymphoblastic leukemia; Philadelphia chromosome-positive chronic myeloid leukemia; Xlinked lymphoproliferative syndrome; neuroblastoma; severe combined immunodeficiency disease; hemophilia; osteogenesis imperfecta; skin reconstruction; acute fatty liver of pregnancy; neurosensory hypoacusis; malaria; and HIV. They have also been applied to treat inborn errors of metabolism, including Gaucher’s disease, Fabry’s disease, fucosidosis, Hurler’s syndrome, metachromatic leucodystrophy, Hunter’s syndrome, glycogenosis, Sanfilippo’s syndrome, Morquio syndrome type B and Niemann-Pick disease. Experimentally, fetal cell and organ transplantation have been studied in an ever-expanding array of diseases. In utero hematopoietic stem cell transplantation is a promising approach for the treatment of a variety of genetic disorders. The rationale is to take advantage of prenatal hematopoietic and immunological ontogeny to facilitate allogeneic hematopoietic engraftment [15e19]. It is an entirely non-myeloablative approach to achieve mixed hematopoietic chimerism and associated donor-specific tolerance. Nonetheless, actual fetal tissue engineering as a therapeutic means has barely begun to be explored, with comparatively few studies being undertaken thus far [8,9,20e44].

FETAL TISSUE ENGINEERING Vacanti and coworkers were the first to make use of fetal cells in engineered constructs, in the late 1980s [6]. The experiment, in rats, used fetal cells from the liver, intestine and pancreas, which were cultured, seeded on bioabsorbable matrices and later implanted. The fetal constructs were implanted in heterologous fashion and heterotopically, namely in the interscapular fat, omentum, and mesentery, with no structural replacement. They were removed for histological analysis no later than two weeks after implantation. Successful engraftment was observed in some animals that received hepatic and intestinal constructs, but in none that received pancreatic ones. 514

Only in 1995 was a second study performed, by the same group, involving fetal liver constructs, also implanted in heterologous and heterotopic fashion in rats [7]. Then, fetal hepatocytes were shown to proliferate to a greater extent than adult ones in culture and to yield higher cross-sectional cell area at the implant. As in the first experiment, neither structural replacement, nor functional studies were included. Fetal constructs as a means of structural and functional replacement, in autologous fashion, in large animal models, were first reported experimentally in 1997 [8,9]. Those studies introduced a novel concept in perinatal surgery, involving the minimally invasive harvest of fetal cells, which are then used to engineer tissue in vitro in parallel to the remainder of gestation, so that an infant, or a fetus, with a prenatally diagnosed birth defect can benefit from having autologous, expanded tissue readily available for surgical implantation, in the neonatal period or before birth.

Congenital anomalies Congenital anomalies are present in 3e4% of all newborns. Those diseases are responsible for nearly 20% of deaths occurring in the neonatal period and even higher morbidity rates during childhood. By definition, birth defects entail loss and/or malformation of tissues or organs. Treatment of many of those congenital anomalies is often hindered by the scarce availability of normal tissues or organs, either in autologous or allologous fashion, mainly at birth. Autologous grafting is frequently not an option in newborns due to donor site size limitations, and the well known severe donor shortage observed in practically all areas of transplantation is even more critical during the neonatal period. Although yet to be fully explored, several studies utilizing fetal tissue engineering as a means to treat congenital anomalies have already been reported experimentally [8,9,20e44]. So far, these studies have involved different models of congenital anomalies/structural replacements,

CHAPTER 26 Tissue Engineering and Transplantation in the Fetus

involving the skin, bladder, trachea, diaphragm, myocardium, heart valves, blood vessels, chest wall, and craniofacial structures. However, many other anomalies are likely to benefit from this therapeutic principle. The first clinical application of fetal tissue engineering is expected in the near future, pending arguably more stringent regulatory clearances, as perhaps expected for a concept that involves both the mother and the infant, as well as unique immature cells.

Alternative sources of fetal cells Fetal cells amenable to processing for tissue engineering can be obtained from a variety of sources besides the fetus. These include the amniotic fluid and membrane, placenta, Wharton’s jelly, and umbilical cord blood. Although peripheral maternal blood can be a source of fetal cells, their consistent isolation in numbers and phenotypes compatible with tissue engineering remains to be demonstrated. So far, of all these sources, the amniotic fluid and the placenta have been the most appealing clinically, to a large extent because they are the least invasive ones for both the mother and the fetus, also because amniocentesis and chorionic villus sampling are widely accepted forms of prenatal diagnostic screening. This is particularly true for the amniotic fluid, in that a diagnostic amniocentesis is routinely offered to any mother with a fetus in whom a structural anomaly has been diagnosed by prenatal imaging (Fig. 26.1).

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FIGURE 26.1 Diagram representing the concept of fetal tissue engineering from amniotic fluid cells for the treatment of birth defects. A small aliquot of amniotic fluid is obtained from a routine amniocentesis, typically performed when a structural anomaly is diagnosed by routine prenatal imaging screening. Fetal tissue is then engineered in vitro from amniotic progenitor cells while pregnancy continues, so that the newborn, or a fetus, can benefit from having autologous, expanded tissue promptly available for surgical reconstruction at birth or in utero.

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This eliminates any additional risk to the mother and thus expands the ethically sensible range of fetal tissue engineering as a form of perinatal therapy beyond life-threatening structural anomalies. Many amniotic and placental cells share a common origin: the inner cell mass of the morula, which gives rise to the embryo itself, the yolk sac, the mesenchymal core of the chorionic villi, the chorion, and the amnion. Most, if not all types of progenitor cells that can be isolated from the amniotic fluid and the placenta seem to share many characteristics [45]. However, a common origin of amniotic and placental progenitor cells remains to be unequivocally demonstrated. The full spectrum of cell types that can be obtained from amniotic and placental progenitor cells still has yet to be defined [38]. Moreover, certain fetal pathologic states, such as neural tube and body wall defects, among others, may lead to the availability of cells not normally found in healthy pregnancies, which could become clinically useful [46].

AMNIOTIC FLUID Embryonic and fetal cells from all three germ layers have long been identified in the amniotic fluid [47e50]. However, the specific origins of many subsets of these cell populations still remain to be determined. The cellular profile of the amniotic fluid varies with gestational age [51]. In addition to the common origin with the mesenchymal portion of the placenta, as mentioned above, the amniotic cavity/fluid receives cells shed from the fetus and, quite possibly, from the placenta as well (although the latter is yet to be definitely confirmed).

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The mechanisms responsible for the production and turnover of the amniotic fluid are thought to also determine the cell types present in the amniotic cavity. In the first half of gestation, most of the amniotic fluid derives from active sodium and chloride transport across the amniotic membrane and fetal skin, with concomitant passive movement of water. In the second half, most of the fluid comes from fetal micturition. An additional substantial source of amniotic fluid is secretion from the respiratory tract. Fetal swallowing and gastrointestinal tract excretions, while not voluminous, also play a role in the composition of the amniotic fluid. As a result of such fluid dynamics, cells present in the urinary, respiratory, and gastrointestinal tracts are shed into the amniotic cavity. Overall, amniotic fluid composition changes predictably throughout gestation. In humans, it is isotonic with fetal plasma in early pregnancy, due to transudation of fetal plasma through the maternal deciduas, or through the fetal skin prior to keratinization, which occurs at approximately 24 weeks. Afterwards and until term, it becomes increasingly hypotonic relative to maternal or fetal plasma. All the variables that play a role in amniotic fluid composition seem to contribute to the changeable profile of its cellular component [51]. Still, much remains to be clarified about the ontogeny of many subsets of amniocytes at any gestational age [45]. This is particularly true before the 12th week of gestation due, to a large extent, to the limitations of performing amniocentesis before that time. The fact that certain progenitor cells can be found in the amniotic fluid was apparently first reported in 1993, when small, nucleated, round cells identified as hematopoietic progenitor cells were found therein (only) before the 12th week of gestation, possibly coming from the yolk sac [52]. A study from 1996 was the first to suggest the possibility of mutilineage potential of non-hematopoietic cells present in the amniotic fluid, by demonstrating myogenic conversion of amniocytes [53]. That study did not specify the identity of the cells that responded to the myogenic culture conditions, in that case the supernatant of a rhabdomyosarcoma cell line. The presence of mesenchymal cells in the amniotic fluid has been proposed for decades [54,55]. However, the differentiation potential of aMSCs started to be determined only fairly recently [34,37,56e60]. Likewise, the presence of possibly more primitive, embryonic-like stem cells in the amniotic fluid was suggested only in the last few years [59,61e64].

CHAPTER 26 Tissue Engineering and Transplantation in the Fetus

Human amniotic epithelial cells have shown pluripotency, being able to differentiate at least into neural and glial cells, and into hepatocyte precursors. These cells have been employed therapeutically in animal models of cerebral ischemia and spinal cord injury, and as experimental transgene carriers into the liver. However, they are not yet universally considered amniotic fluid cells and will not be further discussed here. Also, given the other, more practical sources of fetal hematopoietic stem cells, other than the difficult pre-12th week amniocentesis, it is unlikely that the amniotic fluid will be a useful option for a source of these cells in clinical practice. Hence, this overview will focus on the mesenchymal and embryonic-like stem cells.

aMSCs The amniotic fluid is rich in MSCs. We have described a very simple protocol for isolation of these aMSCs, based on mechanical separation and natural selection by the culture medium [28,30,65]. Other protocols for isolating aMSCs have also been described [55,58]. Previous data on amniotic cell culture, without description of the specific nature of the cells grown (possibly predominantly mesenchymal), show that low oxygen tension in the gas phase can be an effective means of enhancing clonal cell expansion. Although not routinely employed, if necessary, molecular HLA typing can be performed on DNA obtained from expanded MSCs, as well as from fetal and maternal blood cells, by polymerase chain reaction/sequence-specific oligonucleotide using a reverse dot blot method, in order to confirm the fetal origin of the cultured cells. The precise origin(s) of the MSCs found in the amniotic fluid remains to be determined. At first, these cells were thought to be simply shed by the fetus at the end of their life cycle. However, they may actually come from the fetus itself, and/or the placenta, and/or the inner cell mass of the morula, staying viable within the fluid. In no way are aMSCs at the end of their life cycle. Ovine data have shown that amniotic fluid-derived MSCs (aMSCs) proliferate significantly faster in culture than immunocytochemically comparable cells derived from fetal or adult subcutaneous connective tissue, neonatal bone marrow, and umbilical cord blood [28,66]. In humans, the expansion potential of aMSCs exceeds that of bone marrow MSCs [30,57,65]. The phenotype of human aMSCs expanded in vitro is similar to that reported for MSCs derived from second-trimester fetal tissue and adult bone marrow [57,66e68]. Human aMSCs have shown not only mesodermal differentiation potential at least into fibroblasts, adipocytes, chondrocytes, osteocytes, and myogenic lineages, after exposure to specific culture conditions (Figs. 26.2 and 26.3) [30,34,57,68,69]. The progenitor nature of these cells imparts the possibility that they could be used to engineer constructs to correct a wide variety of defects. For example, recent large animal studies have shown that the repair of congenital diaphragmatic hernia, tracheal, chest wall and craniofacial defects can be enhanced by the respective use of tendon, cartilaginous/airway, or bone grafts engineered from aMSCs during surgical reconstruction (Figs. 26.4 and 26.5) [24,32,33,37,39,40,43,44]. Another

FIGURE 26.2 Representative, crosssectional view of a 3D cartilaginous tube engineered from aMSCs (right) seeded onto a poly(lactic-co-glycolic acid) scaffold (left), previously maintained in a bioreactor under chondrogenic conditions.

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FIGURE 26.3 Representative gross view of a 3D osseous construct engineered from aMSCs seeded onto poly-L-lactic acid electrospun nanofibrous scaffolds, previously maintained in a bioreactor under osteogenic conditions.

FIGURE 26.4

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An intact ovine diaphragmatic tendon seen from the chest, 12 months after autologous repair with an engineered, mesenchymal amniocyte-based construct. The dotted line encircles the area of the graft. Reproduced, with permission, from ’Kunisaki, S. M., J. R. Fuchs, A. Kaviani, J. T. Oh, D. A. LaVan, J. P. Vacanti, J. M. Wilson and D. O. Fauza (2006). ’Diaphragmatic repair through fetal tissue engineering: a comparison between mesenchymal amniocyte- and myoblast-based constructs.’ J Pediatr Surg 41(1): 34e9; discussion 34e9’.

proposed use of an engineered construct based on amniotic mesenchymal (and epithelial) cells is for the repair of premature rupture of membranes during pregnancy, but this has yet to be consistently achieved in vivo [70]. An intriguing characteristic of aMSCs is what seems to be unique immunogenic and immunomodulatory properties [71,72]. These cells appear to be immunologically privileged when compared to mesenchymal cells derived from fetal or adult tissue. Their depressed immunogenicity is stable or even further down regulated after multiple passages in culture [71,73]. It has been shown that MSCs enhance the engraftment of umbilical cord blood-derived CD34þ hematopoietic cells [68]. The amniotic fluid has been proposed as a useful source of MSCs to be cotransplanted with hematopoietic stem cells from the same donor [58]. This could be particularly useful in the setting of umbilical cord blood transplantation between siblings. In utero, transamniotic gene transfer has shown some promise in animal models [74,75]. Young, highly proliferative, less differentiated fetal cells are natural targets for the optimization of gene therapy. Thus, amniotic and placental stem cells could certainly make useful targets for genetic manipulation. This is a potentially exciting development for the foreseeable future. More recently, an actual biological role for aMSCs has been uncovered. It has been demonstrated that these cells actually enhance normal fetal wound healing [76]. This germane finding was apparently the first demonstration of a biological role for any amniotic cell. It also provided biological validation for the use of aMSCs in regenerative strategies. Within the

CHAPTER 26 Tissue Engineering and Transplantation in the Fetus

FIGURE 26.5 Representative gross view of a longitudinally opened aMSCengineered tracheal implant at necropsy showing the intraluminal glistening typical of epithelialization, confirmed histologically.

spectrum of reparative and regenerative processes, fetal wound healing is closer to the latter than to the former. When compared with healing at any stage of postnatal life, wound healing in the fetus involves significantly less inflammation and can be almost scarless, particularly early in gestation. That study has added a new facet to fetal and general wound healing, and has also lent biological support to the use of these cells in a spectrum of conceivable regenerative strategies. Finally, the translational relevance of aMSCs in fetal tissue engineering has been underlined by the viability of scaled up manufacturing of human aMSCs in compliance with regulatory guidelines and by the establishment of amniotic cell banks [77,78].

Embryonic-like stem cells From the very little that is currently known about what could be more primitive, embryoniclike stem cells (ESCs) present in the human amniotic fluid, these cells are very scarce, representing zero (i.e., they cannot always be isolated) to less than 1% of the cells present in amniocentesis samples [59,61,62,64]. In addition, these cells have been identified mostly through markers commonly present in ESCs, such as (putatively e see next paragraph) Oct-4, stem cell factor, vimentin, alkaline phosphatase, CD34, CD105, and cKit, but these were not necessarily concomitantly expressed, nor are they exclusive of ESCs proper. Specifically, these markers can also be expressed, alone or in various combinations, in embryonic germ cells; embryonic fibroblasts; embryonal carcinoma cells; MSCs; hematopoietic stem cells; ectodermal, neural, and pancreatic progenitor cells; and fetal and adult nerve tissue; among others [50,67,79e81]. Telomerase activity, another marker for stem cell pluripotency, has also been detected in amniotic fluid cell samples. As to Oct-4 expression, a central component of the characterization of pluripotency and supposedly expressed in these cells; it must be noted that a recent review study has revealed that the previous reports documenting its expression in these cells, and also in aMSCs, warrant careful re-examination with regard to vital technical pitfalls such as gene and protein isoforms, pseudogenes, and antibody choice, as well as primer design [82]. Although specific detection

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of Oct-4A mRNA is achievable, an antibody that can reliably distinguish between Oct-4A (the correct isoform of Oct-4 associated with pluripotency and thus tumorigenesis) and the pseudogene Oct-4B actually remains to be described. Indeed, one could say that true pluripotency based on Oct-4A expression in these cells actually remains to be definitely established [82]. This is further underscored by the fact that these cells do not seem to be tumorigenic in vivo. Still, it is well established that markers alone are not considered enough to characterize human ESCs. A uniform and universal differentiation potential needs to be demonstrated, which remains to be verified in these so-called amniotic ESCs. So far, some of the amniotic cells that express the markers mentioned above have been shown to differentiate into muscle, adipogenic, osteogenic, nephrogenic, neural and endothelial cells, but not necessarily from a uniform population of undifferentiated cells. On the other hand, in accordance with an ESC profile, these pluripotent cells seem to be clonogenic, as at least some of them express cyclin A, a cell cycle regulator. Therefore, although the data currently available can indeed be considered promising, final proof that true ESCs can be consistently isolated from the amniotic fluid, and at which gestational ages, remains to be established. Furthermore, multiple evidences of correspondence between what are considered amniotically-derived ESC-like cells and aMSCs point to the likelihood of basic nomenclature overlaps of what could be just one cell type receiving different names. Regardless, in light of the known presence of other, non-mesenchymal and non-ESC-like stem cells in the amniotic fluid, the simple term ’amniotic stem cell’ is thus too vague.

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Different cell types are found in the placenta at different gestational ages, as a result of the mechanisms behind placental development. In humans, placental villous development starts between 12 and 18 days post-conception (p.c.), when the trophoblastic trabeculae of the placental anlage proliferate and form trophoblastic protrusions, the primary villi, into the maternal blood surrounding the trabeculae. Two days later, embryonic connective tissue from the extra-embryonic mesenchyme invades these villi, which then become secondary villi. Between days 18 and 20 p.c., the first fetal capillaries begin to appear in the now abundant mesenchyme of the villous stroma, marking the development of the tertiary villi, the first generation of which are the mesenchymal villi. Mesenchymal villi are the first structures providing the morphological requisites for materno-fetal exchange of gases, nutrients, and waste. They are also the precursors of all other villous types, namely immature intermediate villi, stem villi, mature intermediate villi, and terminal villi. At approximately the fifth week p.c., all placental villi are of the mesenchymal type. From about the 23rd week p.c. until term, the pattern of villous growth changes, with the mesenchymal villi transforming into mature intermediate villi, rather than into immature ones. During that phase, few mesenchymal and immature intermediate villi remain, in the centers of the villous trees, where they comprise a poorly differentiated growth reserve. As shown above, placental villous sprouting entail active growth of both trophoblastic and mesenchymal tissue components in a coordinated fashion. Further differentiation of the mesenchymal villi into immature or mature intermediate villi is a determining factor in the balance between growth and maturation of the placenta, which, in turn, has a direct impact on the cell types that can be isolated from the placenta at different gestational ages. The genetic and molecular mechanisms behind placental development have hardly begun to be clarified and should also have a bearing on the pluripotency of placental cells. Interestingly, against the conventional wisdom of searching for placental-specific genes that would control this process, most of the genes that have been shown to be essential for placental development are also involved in the development of other organs [83]. No more than a very limited set of genes is expressed exclusively in the placenta [83].

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Since much of the placenta comes from the inner cell mass of the morula, the presence of embryonic progenitor cells in the placenta has long been proposed. More specific types of stem cells, such as trophoblastic, hematopoietic, and MSCs have also been identified in the placenta. Trophoblast stem cells, also known as trophoendoderm stem cells, are defined as cells with the potential to give rise to all differentiated trophoblast cell subtypes, as well as to yolk sac phenotypes. Interestingly, one trophoblast subpopulation, the cytotrophoblasts of the basal decidua, may undergo a full transition to mesenchymal phenotype when they infiltrate maternal mesenchymally-derived uterine stroma and arterial walls. At present, trophoblast stem cells have limited, if any, foreseeable potential for clinical application and will not be reviewed here. Placental/umbilical hematopoietic stem cells have already shown high clinical relevance, but can also be isolated from umbilical cord blood and will be reviewed in another chapter. Placental mesenchymal stem cells (pMSCs) and more primitive, ESCs will be further discussed below.

pMSCs As described above, the placenta has a large mesenchymal component. While the role of this component in placental development has yet to be better understood, we already know that it relates, in part, to the pluripotent potential of pMSCs. For example, recruitment of these cells supports the so-called vasculogenesis that occur during vascularization of the villous sprouts, in addition to the angiogenesis based on the proliferation of endothelial precursors. These mesenchymal cells also play other roles in placental development, such as the paracrine signals that they send to control the stability of the cytotrophoblast column, which in turn determines the degree of trophoblast invasiveness. Yet, much about the mesenchymal core of the placenta remains to be elucidated. pMSCs can be isolated by a number of different protocols. We have described an easily reproducible method, analogous to the one described above for the separation of aMSCs (i.e., based on mechanical separation and natural selection by the culture medium), which can be employed in both ’full thickness’ and chorionic villus sampling placental specimens [29,65]. These cells have unique characteristics when compared with other mesenchymal cells. They proliferate more quickly in culture than comparable cells harvested from fetal or adult tissue, at a similar rate to that of aMSCs [29,65]. Also like aMSCs, they are often stained by monoclonal cytokeratin antibodies, which is a rare finding in mature mesenchymal cells, but common in fetal and umbilical stroma, smooth muscle tumors, and stromal cells associated with reactive processes. It is as yet unclear whether this immunoreactivity is a result of cross-reactivity between a common epitope found in smooth muscle cells, myofibroblasts, and intermediate-sized filaments of cytokeratin, or whether it actually indicates cytokeratin expression in non-epithelial cells. In addition to their natural differentiation into smooth muscle cells during placental development, pMSCs have been shown to be able to differentiate into all mesodermal, as well as some neural cells lineages, in vitro [84,85]. Given the many similarities between pMSCs and aMSCs, as shown above, it is reasonable to speculate that these two cell subsets are actually the same. This, however, remains to be unequivocally demonstrated.

Embryonic-like stem cells The embryonary origin of the placenta renders it a natural candidate for a reservoir of ESCs. From the few reported attempts to isolate ESCs from the placenta, like their amniotic counterparts, what could be ESCs are present in zero to less than 1% of the cells present in placental samples. These cells have also been identified mostly through certain markers, including CD34, CD105, and cKit, that, again, were not necessarily concomitantly expressed and are not exclusive of ESCs.

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These pluripotent cells have shown potential for self-renewal, but a uniform and universal differentiation potential of these cells also remains to be verified. Like their amniotic counterparts, thus far some of these cells have been shown to differentiate into a variety of cell types, but not necessarily from a uniform population of undifferentiated cells. Another limitation of the existing data is the fact that the placenta is rich in hematopoietic stem cells, known not to be committed solely to the hematopoietic lineage. In addition to blood cells, they can also give rise, at least, to neurons, hepatocytes, and muscle cells, and might have contributed to the differentiation findings reported to date. Again, although the existing data is promising and in accordance with the origins of the placenta, final proof that true ESCs can be consistently isolated from that organ, and at which gestational ages, remains to be verified. Another interesting potential development of the study of placental cells is the establishment of 3D models of the placenta and of maternal-fetal circulation through the maintenance of live engineered placental models in bioreactors. This could lead to a better understanding of normal and pathological placental physiology, possibly with indirect therapeutic benefits.

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Fetal cells can be documented in the maternal circulation in the majority of human pregnancies. Fetal progenitor cells have been found to persist in the circulation of women for up to decades after childbirth [10]. Of particular interest, a novel population of fetal cells, often termed pregnancy-associated progenitor cells (PAPCs), appears to differentiate in diseased/ injured maternal tissue, apparently providing some degree of local repair. The precise phenotypical identity of these cells, however, remains controversial. They have been considered to be a hematopoietic stem cell, a MSC, or possibly a novel cell type. At this time, peripheral maternal blood has yet to be proven as a viable source of fetal cells in consistently high enough numbers for tissue-engineering applications. This appealing perspective, however, surely deserves continued scrutiny.

ETHICAL CONSIDERATIONS The use of fetal tissue has always been object of intense ethical debate. The main reason for the ethical controversies comes from the fact that the primary source of fetal tissue is induced abortion. Spontaneous abortion usually does not raise many moral issues. The National Institutes of Health, the American Obstetrical and Gynecological Society and the American Fertility Society, in accordance with the provisions that control the use of adult human tissue, have been regulating the use of fetal specimens from this latter source for decades. However, spontaneous abortion generally yields unsuitable fetal tissue, as it is frequently compromised by pathology such as chromosomal abnormalities, infections and/or anoxia. Arguments over the use of fetal tissue from induced abortion are largely based on somewhat limited scientific evidence, along with clashing religious and customary beliefs about the beginning of life. Not surprisingly, despite the efforts of numerous national and international ethical committees and governmental bodies, a consensus has not yet been reached. In the US, in spite (or perhaps because) of an ongoing moratorium on federal funding for fetal tissue transplantation research, an agreement on this issue may be slowly forming, albeit a stable solution should still be years away. This polemic notwithstanding, tissue engineering, as a relatively novel development in fetal tissue processing, adds a new dimension to the discussion concerning the use of fetal tissue for therapeutic or research purposes. If specimens from a live, diseased fetus, or cells from a routine prenatal diagnostic procedure such as an amniocentesis or chorionic villus sampling, are to be used for the engineering of tissue, which in turn is to be implanted in autologous fashion, no ethical objections should be anticipated, as long as the procedure is a valid therapeutic choice for a given perinatal condition. In that scenario, ethical considerations are the very same that apply to any fetal intervention. On the other hand, if fetal-engineered tissue

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is to be implanted in heterologous fashion, ethical issues are analog to the ones involving fetal tissue/organ transplantation, regardless of whether the original specimen comes from a live or deceased fetus, or from banked fetal cells obtained from the amniotic, placenta, or umbilical cord blood. The distinction between autologous and heterologous implantation of engineered fetal tissue is a critical one in that, again, no condemnation of autologous use could be ethically justified. At the same time, regardless of whether an autologous or heterologous application is being considered, should the amniotic fluid or fetal annexes be definitely confirmed as dependable sources of ESCs, the ethical objections to embryo disposal now plaguing the progress of ESC research could possibly be avoided.

THE FETUS AS A TRANSPLANTATION HOST One could envision a number of advantages of a fetus receiving an engineered construct in utero, not only from a theoretical perspective, but also from clinical and experimental evidence derived from intra-uterine cellular transplantation studies already reported. Those potential advantages encompass: induction of graft tolerance in the fetus, due to its immunologic immaturity; induction of donor-specific tolerance in the fetus by concurrent or previous intrauterine transplantation of hematopoietic progenitor cells; a completely sterile environment; the presence of hormones, cytokines and other intercellular signaling factors that may enhance graft survival and development; the unique wound healing properties of the fetus; and early prevention of clinical manifestations of disease, before they can cause irreversible damage. Most of those advantages should be more or less evident, depending on the gestational timing of transplantation.

Fetal immune development Among the potential benefits of in utero transplantation, the singularity of the fetal immune system deserves special attention. In that respect, basic research on fetal development, as well as studies involving pre and postnatal transplantation of lymphohematopoietic fetal cells have contributed to a better understanding of the fetal immune response. Fetal tolerance resulting in permanent chimerism has long been shown to occur in nature in non-identical twins with shared placental circulation [86,87]. Little is known, however, about precisely when and by what mechanism this tolerance is lost. During fetal development, the precursors of the hematopoietic stem cells arise in the yolk sac, migrate to the fetal liver and then to the thymus, spleen and bone marrow. The fetal liver has its highest concentration of hematopoietic stem cells between the 4th and the 20th week of gestation. Because of their cellular immunologic ’immaturity’, the fetal liver and, to a lesser extent, the fetal thymus have been studied as potential sources of hematopoietic stem cells for major histocompatibility complex/incompatible bone marrow transplantation for more than half a century now. Umbilical cord blood has been increasingly employed as a source of hematopoietic stem cells, in both autologous and heterologous applications [88,89]. Lymphocytes capable of eliciting graft versus host disease (GVHD) are found in the thymus by the 14th week of gestation, but not detectable in the liver until the 18th week. Despite considerable numbers of granulocyte-macrophage colony-forming cells, there is an almost complete absence of mature T cells up to the 14th week in human fetal livers. During gestation, while B cell development takes place mostly in the liver, T cell development occurs predominantly in the thymus. This fact is probably the reason why fetal liver cells are immunoincompetent for cell-mediated and T cell-supported humoral reactions, such as graft rejections and GVHD. Thus, in principle, tissue matching is not necessary in fetal liver transplantation, if this is harvested up to a certain point in gestation. In a number of animal models and small clinical series, fetal liver cells have induced no or merely moderate GVHD in

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histoincompatible donor/recipient pairs. More recently, unique sub-populations of innate lymphoid cells (ILCs), a family of effectors and regulators of innate immunity and tissue remodeling, have been isolated from fetal lung and intestine [90]. The significance of this finding and its potential translational implications has only started to be explored. Although fetal liver stem cells should not cause GVHD, they could still be subject to rejection. Because of this, fetal liver stem cell transplantation has been attempted in the clinical setting preferably in patients with depressed immune function, such as in immunodeficiencies, replacement therapy during bone marrow compromise and during fetal life (in utero transplantation). The same principle applies to the use of fetal thymus. Fatal cases of GVHD are considered much less likely in patients who receive fetal liver stem cells harvested before the 14th week of gestation. This complication, however, has been reported in a patient who received liver cells from a 16-week-old fetus [91]. With umbilical cord blood stem cell transplantation, the incidence of GVHD has been minimal [88,89]. Umbilical cord blood-derived MSCs have also been shown to help prevent (but not treat) GVHD in a rodent model [92]. On the other hand, umbilical cord and placental blood, while rich in hematopoietic progenitor cells, contain alloreactive lymphocytes. Although those lymphocytes are also immature, it is unclear, however, whether they are more or less reactive than adult ones. Compared with those from adult blood, the proportions of activated T cells and helperinducer subsets (CD4/29) are significantly reduced, while the helper-suppressor (CD4/45A) subset is significantly increased [93]. Cord blood natural killer cell activity is low or similar to that in adult blood, but lymphokine-activated killer cell activity may be higher.

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Another germane aspect of intra-uterine transplantation is the fact that maternal cells trafficking into the fetus may pose as the chief barrier to effective engraftment of allogenenic cells or tissues delivered prenatally. It has been recently demonstrated experimentally that there may be macrochimerism of maternal leukocytes in the fetal blood of, with substantial increases in T cell trafficking after intra-uterine transplantation [94,95]. This suggests that the clinical success of intra-uterine transplantation, at least of hematopoietic stem cells, may be improved by transplanting cells also matched to the mother.

APPLICATIONS OF IN UTERO TRANSPLANTATION Cellular intra-uterine transplantation has been employed clinically for decades now, to treat a variety of diseases, including lymphohematopoietic diseases, beta-thalassemia, inborn errors of metabolism and genetic disorders, with some success. Knowledge of the optimal gestational age for transplantation, along with cell selection, route of cell administration and postinterventional tocolysis is still evolving. In utero hematopoietic stem cell transplantation is a non-myeloablative approach to achieving mixed hematopoietic chimerism and associated donor-specific tolerance, improving survival of other grafts later in life. Through prenatal transplantation of hematopoietic progenitor cells, both allogeneic and xenogeneic chimerisms have been induced in animal models and allogeneic chimerism has been achieved in humans [15,96e98]. A recent development in prenatal cell transplantation is neural stem cell delivery to the fetal spinal cord as a means of reversing at least some of the local damage associated with neural tube defects, i.e., different variations of spina bifida [13]. Tolerance of allogeneic intra-uterine implantation of an engineered construct has recently been first demonstrated in an ovine model of fetal tracheal reconstruction with heterologous cartilage engineered from aMSCs [33]. In like manner, tolerance to an airway construct made with autologous aMSCs and a xenologous decellularized matrix has also been shown in a fetal ovine model [43]. Clinically, structural airway anomalies could be justifiably repaired prenatally in select patients with defects so severe that breathing would otherwise be simply impossible at birth. Further potential benefits of prenatal implantation of engineered tissue,

CHAPTER 26 Tissue Engineering and Transplantation in the Fetus

such as for example advantages stemming from the unique wound healing capabilities of the fetus, among other potentially unsuspected ones, are yet to be fully explored.

CONCLUSIONS Fetal tissue engineering may become a preferred perinatal alternative for the treatment of a number of birth defects. Given the recently proven viability of minimally invasive fetal cell sources, such as amniotic fluid, placenta, and umbilical cord blood, the promise of fetal tissue engineering applies to both life-threatening and non-life-threatening anomalies. Fetal progenitor cells from various sources are progressively becoming relevant, if not indispensable tools in research related to stem cells, tissue engineering, gene therapy, and maternal-fetal medicine. Still, much remains to be learned and a variety of evolutionary paths, including unsuspected ones, have yet to be pursued in this relatively new branch of tissue engineering. The whole subfield of in utero implantation of engineered tissue also waits to be fully explored. Fetal tissue engineering shall also benefit from the progress expected for the different aspects of tissue engineering in general. Fertile experimental work from an increasing number of groups has introduced promising novel therapeutic concepts utilizing fetal cells. As long as progress is made in the ethical debate over the use of these cells and their banking, the reach of fetal tissue engineering, nonetheless, will likely go beyond the perinatal period, offering unique therapeutic perspectives for different age groups.

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