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doi:10.1111/jog.12213
J. Obstet. Gynaecol. Res. Vol. 40, No. 2: 360–368, February 2014
Therapeutic potential of placenta-derived stem cells for liver diseases: Current status and perspectives Toshio Miki1 and Brendan Grubbs2 1
Department of Biochemistry and Molecular Biology, and 2Department of Obstetrics and Gynecology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA
Abstract Over the last decade, there has been a growing interest in the human placenta as a unique source of stem cells. The placenta is a fetal organ that is normally discarded following delivery. Therefore, it is readily available as a source of cells without the ethical concerns normally associated with embryonic stem cells. These cells also carry less risk for age- and environmental-related DNA damage. In addition to these practical advantages of placenta-derived cells, amniotic epithelial cells possess unique stem cell-like biological characteristics. In contrast to other parts of the placenta, cells from the amniotic epithelium are derived from pluripotent epiblasts and possess the ability to differentiate into all three germ layers. From a translational perspective, amnion-derived stem cells are very attractive candidates for clinical application. These cells are genetically stable and do not demonstrate tumorigenicity upon transplantation, and may be endowed with immunomodulatory and/or anti-inflammatory properties. These unique characteristics have made amniotic epithelial cells attractive for use as stem cell-based therapies for liver disease. Human and rodent amniotic epithelial cells have already demonstrated their therapeutic efficacy in multiple animal models. Although the detailed mechanism by which the transplanted cells generate a therapeutic effect is not yet totally understood, these dramatic results have generated significant interest for consideration of these amnion-derived stem cells for clinical applications. This review covers recent findings of the therapeutic potential of amnion-derived stem cells for liver diseases, and provides perspectives for future developments. Key words: cirrhosis, congenital metabolic disorders, liver, placenta, stem cells.
Introduction The placenta functions throughout fetal life to exchange gas, nutrients, and waste products with the maternal circulation.1 After playing this crucial role, the placenta is generally discarded following delivery. In the USA alone, there are more than 4 million live births per year, making the placenta a readily available source of biological samples without the need for additional invasive procedures. Procurement of these cells, therefore, does not elicit the ethical concerns that accompany the use of embryonic stem cells. In addition, these
neonatal cells exhibit intact host DNA that carries little to no environmentally acquired or age-acquired DNA damage. The placenta consists of two components: the discoid component (disc) and the membranous component.2 The disc is composed of a fetal portion (chorionic plate) and a maternal portion (basal plate, decidua basalis). Between these layers, lies the intervillous space, which is fed by branches of the uterine artery, and drains into the uterine vein. Projecting into this space from the fetal side are the capillary rich chorionic villi, which allow for exchange between the two separate
Received: May 31 2013. Accepted: June 17 2013. Reprint request to: Dr Toshio Miki, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, 1425 San Pablo Street, BCC 412, MC 9080, Los Angeles, CA 90033, USA. Email: [email protected]
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circulations via simple and facilitated diffusion, active transport, and pinocytosis. The chorionic plate is the main body of the placenta, and houses the bulk of placental circulation. It receives blood from the fetus via the umbilical arteries and returns blood to the fetus via the umbilical vein. The membranes are composed of the amnion, the chorion and the decidua capsularis.3 The amnion consists of three layers with two types of cells. The inner most (fetal side) compact layer consists of amniotic epithelial cells attached to a basement membrane. The second layer reinforces the inner layer with mesenchymal stromal cells. The outer layer, called the spongy layer or zona spongiosa, is adjacent to the chorionic membrane and functions to protect the inner layers by absorbing mechanical stress from the outside. Both human amniotic epithelial cells (hAEC) and amniotic mesenchymal stromal cells (hAMSC) have been proposed to possess stem cell-like characteristics.4–6 In this review we provide a brief overview of the stem cell characteristics of these amnion-derived stem cells and focus on summarizing recent studies that have demonstrated therapeutic efficacy of these cells for liver diseases.
Unique Developmental Aspects of Amniotic Epithelial Cells The placenta consists of multiple layers derived from developmentally different origins. Unlike the bulk of the placenta, which is derived from trophectoderm, amniotic epithelial cells are derived from epiblasts. By the 8th day following fertilization, the inner cell mass (ICM) of the blastocyst differentiates into two layers, the hypoblast and the epiblast. The amniotic cavity develops within the epiblasts, and cells lining the cavity are termed amnioblasts, which eventually develop into amniotic epithelia (Fig. 1). The derivation of amnioblasts from epiblasts occurs about a week prior to gastrulation, which is the point at which the fate of most somatic cells is specified. The cells are further spatially segregated throughout gestation from the epicenter of organogenesis (embryo/fetus) by amniotic fluid. Therefore, it is speculated that the epiblast-derived amnioblasts do not receive differentiation signals in a systemic fashion. In spite of the rapid expansion of the amniotic membrane surface, the mitotic division rate of AEC is relatively low, peaking around day 20 (0.8%) and stopping by day 180.7
Figure 1 Developmental origin of amnion-derived stem cells. Schematic structure of early stage of human embryo (left) and hematoxylin–eosin staining of amnion in human term placenta (right) showing developmental origin of both human amniotic epithelial cells and amniotic mesenchymal stroma cells. The amnioblast (green) derived from pluripotent epiblasts at day 8 after fertilization. Concomitantly, extra-embryonic mesoblasts (red) arise and cover the interior of the cytotrophoblast and the exterior of the primary yolk sac as well as the amniotic cavity.
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The unique developmental environment of amniotic epithelial cells leads to the hypothesis that some amniotic epithelial cells may maintain epiblast-like characteristics throughout gestation until after the delivery.8 Indeed, embryonic stem cell surface markers are still expressed on a population of amniotic epithelial cells in term placentae.9,10 These include stage-specific embryonic antigen-3 (SSEA-3), tumor rejection antigen 1–60 (TRA1-60), and TRA1-81, which are expressed on 5–10% of hAEC. Furthermore, the number of stem cell marker positive cells is higher in the fetal amnion at 18–19 weeks (30–40%) than at term, indicating that the number of stem cells of epiblast origin stochastically decreases as pregnancy progresses.11 Taken together, these observations indicate that most of the AE cells spontaneously differentiate, and amniotic epithelium in term placenta contains cells in various stages of development. It is not yet clarified if this spontaneous differentiation is towards a specific single lineage or multiple lineages. Several groups have demonstrated the developmental potential of hAEC.9,12 Primary hAEC differentiate into cells of all three germ layers under appropriate culture conditions (Table 1). Although these data indicate that hAEC likely possess multipotency, this has not yet been proven at the single cell level. If the spontaneous differentiation of amnioblasts has occurred and this differentiation occurred randomly towards all three germ cell lineages, the primary hAEC population in term placenta contains all lineage-committed cells as well as stem cells. Each culture condition that has been used in order to induce in vitro differentiation might instead select particular lineage committed cells. Further studies must be conducted to clarify the pluri-/multipotency of a single hAEC. Table 1 Developmental potential of amnion-derived stem cells
AEC
AMSC
Lineage
Types of target cells
References
Ectoderm Mesoderm Endoderm
Neuron, glial cell Cardiomyocyte β-cells, hepatocyte
9,12
Ectoderm Mesoderm
Neuron Adipocyte, chondrocyte, osteocyte, skeletal myocyte, cardiomyocyte, endothelial cell β-cells, hepatocyte
19–21
Endoderm
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9,12,13 9,12,14–18
21–26
19,27
Origin of Amniotic Mesenchymal Stromal Cells When the amniotic cavity has developed, extraembryonic mesoblasts arise and line the interior of the cytotrophoblast, the exterior of the primary yolk sac, and the amniotic cavity. It is unclear if the extraembryonic mesoblasts are derived from the trophoblast or the epiblast.28 The extra-embryonic mesoblasts eventually develop into the amniotic connective tissue, including the AMSC (Fig. 1). As other mesenchymal stem cells, hAMSC are defined as plastic-adherent cells that can be subcultured several times, and display a specific cell surface antigen profile. Freshly isolated hAMSC express CD13, CD29, CD44, CD54, CD73, CD105, CD166 and the absence of CD34, CD45, CD14, and CD31. hAMSC demonstrate mesenchymal stem cell-specific differentiation potential into osteocytic, chondrocytic and adipocytic mesodermal lineages.19–25 In addition, studies have reported that hAMSC also possess differentiation potential toward neural (ectoderm),20,21 skeletal muscle, cardiomyocitic, endothelial (mesoderm),23,24 pancreatic, and hepatocytic (endoderm)19,27 lineages. In these reports, however, tested hAMSC were not carefully purified from the hAEC population. These non-mesodermal differentiation potentials might actually be provided by the contaminated lineage committed hAEC described above.
Immunomodulatory Properties of Amnion-derived Cells It is well recognized that amniocytes express low levels of MHC class I, which leads to low immunogenicity.29,30 The recent finding of expression of HLA-G and costimulatory molecules CD40L, CD80, CD86 and PD-1 also contributes to the observed immuneprivileged status of amnion-derived cells.13,26,31–34 These cells also produce and release Fas ligand, which further serves an immunomodulatory function. It has been reported by several groups that hAEC and hAMSC inhibit the allogeneic mixed lymphocyte reaction (MLR) in a dose-dependent manner35–37 as well as proliferation of T- and B-lymphocytes. Other studies have indicated that various soluble factors produced by amniocytes affect the T-cell response.38 Suggested potential humoral factors are tumor necrosis factor (TNF)-α Fas ligand (FasL), TNF-related apoptosisinducing ligand (TRAIL), transforming growth factor (TGF)-β,38 indoleamine 2,3-dioxygenase (IDO), and interleukin-10 (IL-10).39–41
© 2013 The Authors Journal of Obstetrics and Gynaecology Research © 2013 Japan Society of Obstetrics and Gynecology
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As the amnion layer is separated from the fetomaternal interface by the chorion, it is questionable if these unique immunomodulatory properties contribute to maintaining fetomaternal tolerance during pregnancy, and is instead due to the immaturity of these cells. Regardless of the role for these properties in nature, they provide a remarkable advantage for their use in cell therapy-based treatments.
Lack of Stem Cell Tumorigenicity Tumorigenicity of stem cells represents the key obstacle for safe clinical use of cell-based regenerative medicine therapies. Unlike ES cells, AE cells are not tumorigenic upon transplantation. In the early 1980s, human amnion was transplanted into patients in order to either test immunogenicity29 or to restore a glycogen deficiency.42–45 No tumor formation was reported in these clinical transplant cases. In addition to these clinical data, Miki et al. tested the tumorigenicity of hAEC using immunodeficient SCID/Beige mice.9 After observation throughout the lifespan of the recipient animals (∼2 years), no tumor formation was observed. This finding was subsequently confirmed by Ilancheran et al.12 This unique property may be due to the epigenetic state of these cells. Easley et al. conducted epigenetic profiling assays with the hAEC,46 and found that the global DNA methylation status of hAEC was intermediate between pluripotent stem cells (ES cells) and somatic cells (skin fibroblasts) while the histone acetylation status was similar to the somatic cells. This epigenetic modification status could explain the dichotomous property of hAEC that possess both stem cell-like plasticity (differentiation capability) and genetic stability (non-tumorigenic). These data clearly indicate the advantage for the potential clinical use of hAEC.
Hepatic Differentiation Potential of Amnion-derived Stem Cells The potential for hepatic differentiation of hAEC has been demonstrated in multiple studies. In the 1990s Sakuragawa’s group sought to take advantage of the low immunogenicity of hAEC for the purpose of utilizing these cells as gene carriers for the conduction of gene therapy.14 hAEC genetically labeled with the β-galactosidase gene were transplanted into the livers of SCID mice. Interestingly, the hAEC had integrated into the liver parenchyma and expressed both
α-fetoprotein and albumin at 1 and 2 weeks after cell injection. Takashima et al. conducted gene expression analysis on isolated hAEC and demonstrated that some, but not all hepatocyte marker genes were expressed following 2 weeks of culture in media supplemented with hepatocyte growth factor, fibroblast growth factor-2, and oncostatin M.15 Miki et al. identified embryonic stem cell marker positive cells in hAEC and demonstrated in vitro hepatic differentiation using a simple two-step protocol.9,16,17 Ilancheran et al. independently validated these results in 2007.12 In 2011, Marongiu et al. demonstrated that co-culture with mouse hepatocytes induces hepatic maturation in hAEC.18 Interestingly they also demonstrated that activin-A pretreatment, which is an essential step to induce endoderm specification in pluripotent stem cells, was not required for hepatic differentiation of hAEC. This result suggests that some of the hAEC were already committed to an endodermal lineage prior to the hepatic differentiation. As mentioned above, primary hAEC populations may contain both stem cell-like pluri/multi potent cells and endoderm lineage committed cells, with varying degrees of multipotency and lineage commitment. An efficient strategy for isolation of hepatic cells is to first induce in vitro hepatic differentiation in all primary AEC of a population, and afterwards sort out successfully derived hepatocyte-like cells for subsequent transplantation.
Strategy of Stem Cell Technology for Treating Liver Diseases End-stage liver failure is a devastating health-care problem worldwide. The World Health Organization estimates that approximately 10% of the world population, including 25 million Americans, has chronic liver disease. In the USA alone, over 40 000 patients progress to end-stage liver failure and death due to fulminant liver failure. The only effective treatment intervention at this stage is liver transplantation. However, fewer than 6000 liver transplants are performed annually in the USA, resulting in significant rates of waiting list morbidity and mortality. One option to bridge the gap between liver failure and liver transplant is hepatocyte transplantation.47,48 Hepatocyte transplantation (HTx) is a less invasive procedure than whole liver transplant, with less associated morbidity, fewer complications, a significantly reduced recovery time, and a lower cost. More than 50
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cases of clinical HTx have been conducted and have demonstrated therapeutic efficacy.49–53 This cell replacement approach is particularly suitable to treat congenital liver disorders as metabolic disease patients require only partial replacement of hepatocytes to compensate for the particular missing enzyme function.54 In a patient with Crigler–Najjar syndrome, an infusion of normal hepatocytes, equivalent to 5% of the parenchymal mass, was able to achieve a medium-term reduction in serum bilirubin.55 Similarly, a child effected by ornithine transcarbamylase deficiency who received 1.9x109 hepatocytes demonstrated normalization of plasma ammonia and glutamine levels on a normal diet without phenylbutyrate/phenylacetate therapy.56 These cases clearly demonstrate that congenital metabolic disorders can be effectively treated via cell replacement therapy. Currently, the shortage of donor hepatocytes prohibits extensive clinical application of HTx, making stem cell-derived hepatocytes an attractive alternative source.57 Embryonic stem cells and induced pluripotent stem cells are promising sources for the production of hepatic cells, and numerous studies have demonstrated the hepatic differentiation potential of pluripotent stem cells in vitro. The intrinsic risk of teratoma formation and the genetically unstable nature of these cells prevent clinical applications.58 As described above, hAEC possess hepatic differentiation ability and do not form tumors upon transplantation, making them perhaps the most clinically applicable stem cell-derived hepatic cells for this purpose.
Congenital Liver Metabolic Disorders Approximately 1 in 1500 children is born with a congenital metabolic disease (CMD).59 Many of these critical inborn errors of metabolic or synthetic processes principally involve the liver. Current therapy for CMD consists of life-long dietary restrictions with or without supplementation of amino acids. Several preclinical studies using disease model mice provide promising evidence regarding the therapeutic potential of hAEC transplantation and will be summarized below.
Maple Syrup Urine Disease Maple syrup urine disease (MSUD) is a genetic disorder caused by a deficiency of branched-chain alphaketo acid dehydrogenase complex due to mutations in the BCKDHA, BCKDHB, DBT, or DLD genes.60 These
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mutations impair the oxidative decarboxylation of branched-chain keto acids derived from branchedchain amino acids (BCAA). As a result, BCAA, leucine, isoleucine and valine as well as their byproducts accumulate and lead to severe organ damage. The current treatment, lifelong dietary restriction of protein and BCAA, has yielded largely unsatisfactory results, suggesting that new approaches to treat this disease are required. In 2006, Homanics et al. produced an intermediate MSUD (iMSUD) disease model mouse with relevant similarities to the human disease.61 Unlike classic MSUD model mice, disease onset is delayed in the iMSUD mice.62 Skvorak et al. transplanted primary hAEC directly into the liver of neonatal iMSUD mice,63 which significantly improved survival of recipient animals at 35 days (100%) and 100 days (81.2% survival). All untreated control animals died before 30 days of age. The bodyweight of hAEC-recipient mice was normalized along with the restoration of general health. BCAA levels were also significantly improved in the serum and in the brain. Although engrafted hAEC in the recipient liver were not present in the observed histologic sections, expression levels of human BCKDHA, BCKDHB, and DLD mRNA in the liver at 100 days indicate hAEC had engrafted and were enzymatically active in the recipient mouse liver for more than 3 months. This preclinical study demonstrated the remarkable potential of hAEC transplantation. Further studies with clinically applicable grade hAEC are eagerly anticipated to lead to clinical trials.
Niemann–Pick Disease Niemann–Pick disease (Type C1) is also a genetic disorder caused by a mutation of a gene (NPC1) that contributes to intracellular transport of cholesterol and lipids.64 This resulting sphingomyelinase deficiency leads to an accumulation of excess intracellular lipids and leads to both severe liver damage and neurodegeneration. As the pathological condition is systemic, the therapeutic concept of hepatocyte transplantation at first glance does not appear suitable for the treatment of this disease. Hong et al., however, utilized hAEC as a source of enzyme replacement rather than cell replacement in a mouse model of Niemann– Pick disease.65 They injected half a million hAEC every other week from 5 weeks of age. This treatment significantly extended the lifespan of the test group, and resulted in alleviation of tissue damage. The same concept was tested in 1987 by the implantation of human amnion (membrane) into a subcutaneous
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thoracic pouch under the armpit of a 14-year-old Niemann–Pick disease type B patient.42 A total of six implantations were carried out at 1–4-month intervals. Immediately after the first implantation, clinical improvements were observed. Serum linoleic acid level was also significantly increased within 6 months. The therapy was stopped when the subcutaneous pouch could no longer tolerate further implantations. In 1992, Bembi et al. reported on five type B patients in which hAEC were implanted in order to supply exogenous sphingomyelinase.66 After 4 months, the sphingomyelinase activity achieved levels seen in heterozygotes. In two cases, a single implantation normalized urinal sphingomyelin and phospholipid levels. If longterm survival of hAEC can be achieved, these promising results suggest the potential of future therapy for Niemann–Pick disease utilizing hAEC.
Liver Cirrhosis Liver cirrhosis is the end-point of liver fibrosis in many chronic liver conditions, including viral infections, alcohol abuse, drug/chemical administrations, and exposure to radiotherapy. Cirrhosis and chronic liver disease were the 10th leading causes of death for men and the 12th for women in the USA in 2001.67 Cirrhosis occurs when scar tissue replaces the normal liver parenchyma, which impairs function, and blocks normal blood flow through the organ. The cirrhotic liver is not a suitable environment for cell transplantation, regardless of which cell type is used. If cell replacement therapy is conducted before end-stage cirrhosis, however, cell therapy may delay or prevent disease progression. Indeed, some preclinical studies indicate that amnion-derived cell transplantation improves liver fibrosis in animal models. Manuelpillai et al. systemically (via tail vein) transplanted hAEC to a carbon tetrachloride (CCL4)-treated liver cirrhosis mouse model.68 The biochemical marker of liver damage (alanine aminotransferase) significantly improved and hepatocyte apoptosis was reduced after 2 weeks of hAEC transplantation. The study also demonstrated the reduction of hepatic stellate cell activation and hepatic fibrosis. Although direct evidence was not presented, it suggested an increased matrix metalloproteinase (MMP)-2 activity as the mechanism underlying the fibrosis regression. Zhang et al. transplanted human amnion-derived cells into CCL4treated mouse liver via the spleen.69 They also demonstrated an improved ALT score, and a decrease of both apoptosis and fibrosis. Although the amnion-
derived cells were originally hAEC, it appeared after in vitro expansion that the mesenchymal cell population was the dominant cell population. The mechanism of this anti-fibrosis effect should therefore not be considered a property of hAEC, rather more likely a general mesenchymal stem cell property. Manuelpillai followed up the previous study and proposed a novel anti-fibrosis mechanism of hAEC transplantation.70 They demonstrated that transplanted hAEC activate M2 (healing) macrophages rather than M1 (proinflammatory)-type macrophages. Anti-fibrotic effects of amnion-derived cells have also been observed by covering the liver surface with human amniotic membrane. Ricci et al. patched fresh or cryopreserved human amnion membrane onto the surface of rat livers with induced fibrosis, and evaluated the number of α-smooth muscle actin, Desmin, and CK19-positive cells in the treated liver.71 The number of fibroblast marker positive cells was decreased in the amnion-patched group, on both the surface and in the core of the liver. In the follow-up studies conducted by the same group, the researchers concluded that prostaglandins are the key effectors of the immunomodulatory activity of amniotic membrane.72 In summary, transplanted hAEC appear to function in the treatment of cirrhosis by multiple routes, including: secretion of MMP that induce fibrinolysis, inactivation of hepatic stellate cells by a decrease in TGFβ-1 and an increase in IL-10 concentration, and activation of M2 type macrophages. These studies have provided important data; however, they did not conclusively demonstrate these pathways and the interactions of each mechanism. They also did not elucidate the crosstalk between the engrafted hAEC and the recipient’s immune cells. More detailed studies are needed to explain these fundamental questions.
Conclusion In this review we have briefly introduced the hypotheses explaining why AEC, like stem cells, demonstrate developmental potential to all three germ layers.8 We have also focused on potential clinical applications of amnion-derived cells for the treatment of liver diseases, an area in need of novel therapeutic approaches. As summarized here, preclinical studies with disease model animals have successfully demonstrated the therapeutic potential of hAEC transplantation. However, there are still many issues that must be clarified before these cells can be used for the treatment of
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patients. Clinically applicable cells must demonstrate scalability, reproducibility, and purity. Further studies are essential to identify the cell surface markers required for isolation of a pure population, as well as culture conditions that enable expansion of cell numbers without altering cell characteristics. The immunomodulatory effects of hAEC must be comprehensively characterized both in the immediate period following transplantation, and long-term. Amnionderived stem cells possess advantages due to their unique developmental origin. The amnion-derived cells are readily available, pluripotent/multipotent cells that are generated from an ethically acceptable tissue. We must continue accumulating scientific evidence to safely utilize the therapeutic potential of hAEC.
Acknowledgments T.M. is supported by California Institute for Regenerative Medicine (CIRM) grant TR3-05488.
Disclosure The authors have no financial support or relationship that may pose conflict of interest.
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28. Luckett WP. Origin and differentiation of the yolk sac and extraembryonic mesoderm in presomite human and rhesus monkey embryos. Am J Anat 1978; 152: 59–97. 29. Akle CA, Adinolfi M, Welsh KI, Leibowitz S, McColl I. Immunogenicity of human amniotic epithelial cells after transplantation into volunteers. Lancet 1981; 2: 1003–1005. 30. Sakuragawa N, Tohyama J, Yamamoto H. Immunostaining of human amniotic epithelial cells: Possible use as a transgene carrier in gene therapy for inborn errors of metabolism. Cell Transplant 1995; 4: 343–346. 31. Hammer A, Hutter H, Blaschitz A et al. Amnion epithelial cells, in contrast to trophoblast cells, express all classical HLA class I molecules together with HLA-G. Am J Reprod Immunol 1997; 37: 161–171. 32. Houlihan JM, Biro PA, Harper HM, Jenkinson HJ, Holmes CH. The human amnion is a site of MHC class Ib expression: Evidence for the expression of HLA-E and HLA-G. J Immunol 1995; 154: 5665–5674. 33. Kubo M, Sonoda Y, Muramatsu R, Usui M. Immunogenicity of human amniotic membrane in experimental xenotransplantation. Invest Ophthalmol Vis Sci 2001; 42: 1539– 1546. 34. Pratama G, Vaghjiani V, Tee JY et al. Changes in culture expanded human amniotic epithelial cells: Implications for potential therapeutic applications. PLoS ONE 2011; 6: e26136. 35. Bailo M, Soncini M, Vertua E et al. Engraftment potential of human amnion and chorion cells derived from term placenta. Transplantation 2004; 78: 1439–1448. 36. Wolbank S, Peterbauer A, Fahrner M et al. Dose-dependent immunomodulatory effect of human stem cells from amniotic membrane: A comparison with human mesenchymal stem cells from adipose tissue. Tissue Eng 2007; 13: 1173–1183. 37. Banas RA, Trumpower C, Bentlejewski C, Marshall V, Sing G, Zeevi A. Immunogenicity and immunomodulatory effects of amnion-derived multipotent progenitor cells. Hum Immunol 2008; 69: 321–328. 38. Li H, Niederkorn JY, Neelam S et al. Immunosuppressive factors secreted by human amniotic epithelial cells. Invest Ophthalmol Vis Sci 2005; 46: 900–907. 39. Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 2005; 105: 1815–1822. 40. Talmi YP, Sigler L, Inge E, Finkelstein Y, Zohar Y. Antibacterial properties of human amniotic membranes. Placenta 1991; 12: 285–288. 41. Buhimschi IA, Jabr M, Buhimschi CS, Petkova AP, Weiner CP, Saed GM. The novel antimicrobial peptide beta3-defensin is produced by the amnion: A possible role of the fetal membranes in innate immunity of the amniotic cavity. Am J Obstet Gynecol 2004; 191: 1678–1687. 42. Scaggiante B, Pineschi A, Sustersich M, Andolina M, Agosti E, Romeo D. Successful therapy of Niemann-Pick disease by implantation of human amniotic membrane. Transplantation 1987; 44: 59–61. 43. Tylki-Szymanska A, Maciejko D, Kidawa M, Jablonska-Budaj U, Czartoryska B. Amniotic tissue transplantation as a trial of treatment in some lysosomal storage diseases. J Inherit Metab Dis 1985; 8: 101–104. 44. Yeager AM, Singer HS, Buck JR et al. A therapeutic trial of amniotic epithelial cell implantation in patients with lysosomal storage diseases. Am J Med Genet 1985; 22: 347–355.
45. Sakuragawa N, Yoshikawa H, Sasaki M. Amniotic tissue transplantation: Clinical and biochemical evaluations for some lysosomal storage diseases. Brain Dev 1992; 14: 7–11. 46. Easley CA 4th, Miki T, Castro CA et al. Human amniotic epithelial cells are reprogrammed more efficiently by induced pluripotency than adult fibroblasts. Cell Reprogram 2012; 14: 193–203. 47. Strom SC, Fisher RA, Rubinstein WS et al. Transplantation of human hepatocytes. Transplant Proc 1997; 29: 2103–2106. 48. Strom SC, Fisher RA, Thompson MT et al. Hepatocyte transplantation as a bridge to orthotopic liver transplantation in terminal liver failure. Transplantation 1997; 63: 559–569. 49. Strom SC, Chowdhury JR, Fox IJ. Hepatocyte transplantation for the treatment of human disease. Semin Liver Dis 1999; 19: 39–48. 50. Strom SC, Bruzzone P, Cai H et al. Hepatocyte transplantation: Clinical experience and potential for future use. Cell Transplant 2006; 15 (Suppl 1): S105–S110. 51. Ohashi K, Park F, Kay MA. Hepatocyte transplantation: Clinical and experimental application. J Mol Med 2001; 79: 617–630. 52. Malhi H, Gupta S. Hepatocyte transplantation: New horizons and challenges. J Hepatobiliary Pancreat Surg 2001; 8: 40–50. 53. Fox IJ. Transplantation into and inside the liver. Hepatology 2002; 36: 249–251. 54. Fox IJ, Chowdhury JR. Hepatocyte transplantation. Am J Transplant 2004; 4 (Suppl 6): 7–13. 55. Fox IJ, Chowdhury JR, Kaufman SS et al. Treatment of the Crigler-Najjar syndrome type I with hepatocyte transplantation. N Engl J Med 1998; 338: 1422–1426. 56. Horslen SP, McCowan TC, Goertzen TC et al. Isolated hepatocyte transplantation in an infant with a severe urea cycle disorder. Pediatrics 2003; 111 (6 Pt 1): 1262–1267. 57. Fox IJ, Strom SC. To be or not to be: Generation of hepatocytes from cells outside the liver. Gastroenterology 2008; 134: 878– 881. 58. Ben-David U, Benvenisty N. Analyzing the genomic integrity of stem cells. 2008. 59. Freeze HH. Understanding human glycosylation disorders: Biochemistry leads the charge. J Biol Chem 2013; 288: 6936– 6945. 60. Larsson A, Therrell BL. Newborn screening: The role of the obstetrician. Clin Obstet Gynecol 2002; 45: 697–710; discussion 30–32. 61. Homanics GE, Skvorak K, Ferguson C, Watkins S, Paul HS. Production and characterization of murine models of classic and intermediate maple syrup urine disease. BMC Med Genet 2006; 7: 33. 62. Skvorak KJ. Animal models of maple syrup urine disease. J Inherit Metab Dis 2009; 32: 229–246. 63. Skvorak KJ, Dorko K, Marongiu F et al. Placental stem cell correction of murine intermediate maple syrup urine disease. Hepatology 2013; 57: 1017–1023. 64. Walterfang M, Bonnot O, Mocellin R, Velakoulis D. The neuropsychiatry of inborn errors of metabolism. J Inherit Metab Dis 2013; 36: 687–702. 65. Hong SB, Seo MS, Park SB, Seo YJ, Kim JS, Kang KS. Therapeutic effects of human amniotic epithelial stem cells in Niemann-Pick type C1 mice. Cytotherapy 2012; 14: 630–638. 66. Bembi B, Comelli M, Scaggiante B et al. Treatment of sphingomyelinase deficiency by repeated implantations of amniotic epithelial cells. Am J Med Genet 1992; 44: 527–533.
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67. Anderson RN, Smith BL. Deaths: Leading causes for 2001. Natl Vital Stat Rep 2003; 52: 1–85. 68. Manuelpillai U, Tchongue J, Lourensz D et al. Transplantation of human amnion epithelial cells reduces hepatic fibrosis in immunocompetent CCl-treated mice. Cell Transplant 2010; 19: 1157–1168. 69. Zhang D, Jiang M, Miao D. Transplanted human amniotic membrane-derived mesenchymal stem cells ameliorate carbon tetrachloride-induced liver cirrhosis in mouse. PLoS ONE 2011; 6: e16789. 70. Manuelpillai U, Lourensz D, Vaghjiani V et al. Human amniotic epithelial cell transplantation induces markers of alterna-
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tive macrophage activation and reduces established hepatic fibrosis. PLoS ONE 2012; 7: e38631. 71. Ricci E, Vanosi G, Lindenmair A et al. Anti-fibrotic effects of fresh and cryopreserved human amniotic membrane in a rat liver fibrosis model. Cell Tissue Bank 2012; doi 10.1007/s 10561-012-9337-x. 72. Rossi D, Pianta S, Magatti M, Sedlmayr P, Parolini O. Characterization of the conditioned medium from amniotic membrane cells: Prostaglandins as key effectors of its immunomodulatory activity. PLoS ONE 2012; 7: e46956.
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doi:10.1111/jog.12213
J. Obstet. Gynaecol. Res. Vol. 40, No. 2: 360–368, February 2014
Therapeutic potential of placenta-derived stem cells for liver diseases: Current status and perspectives Toshio Miki1 and Brendan Grubbs2 1
Department of Biochemistry and Molecular Biology, and 2Department of Obstetrics and Gynecology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA
Abstract Over the last decade, there has been a growing interest in the human placenta as a unique source of stem cells. The placenta is a fetal organ that is normally discarded following delivery. Therefore, it is readily available as a source of cells without the ethical concerns normally associated with embryonic stem cells. These cells also carry less risk for age- and environmental-related DNA damage. In addition to these practical advantages of placenta-derived cells, amniotic epithelial cells possess unique stem cell-like biological characteristics. In contrast to other parts of the placenta, cells from the amniotic epithelium are derived from pluripotent epiblasts and possess the ability to differentiate into all three germ layers. From a translational perspective, amnion-derived stem cells are very attractive candidates for clinical application. These cells are genetically stable and do not demonstrate tumorigenicity upon transplantation, and may be endowed with immunomodulatory and/or anti-inflammatory properties. These unique characteristics have made amniotic epithelial cells attractive for use as stem cell-based therapies for liver disease. Human and rodent amniotic epithelial cells have already demonstrated their therapeutic efficacy in multiple animal models. Although the detailed mechanism by which the transplanted cells generate a therapeutic effect is not yet totally understood, these dramatic results have generated significant interest for consideration of these amnion-derived stem cells for clinical applications. This review covers recent findings of the therapeutic potential of amnion-derived stem cells for liver diseases, and provides perspectives for future developments. Key words: cirrhosis, congenital metabolic disorders, liver, placenta, stem cells.
Introduction The placenta functions throughout fetal life to exchange gas, nutrients, and waste products with the maternal circulation.1 After playing this crucial role, the placenta is generally discarded following delivery. In the USA alone, there are more than 4 million live births per year, making the placenta a readily available source of biological samples without the need for additional invasive procedures. Procurement of these cells, therefore, does not elicit the ethical concerns that accompany the use of embryonic stem cells. In addition, these
neonatal cells exhibit intact host DNA that carries little to no environmentally acquired or age-acquired DNA damage. The placenta consists of two components: the discoid component (disc) and the membranous component.2 The disc is composed of a fetal portion (chorionic plate) and a maternal portion (basal plate, decidua basalis). Between these layers, lies the intervillous space, which is fed by branches of the uterine artery, and drains into the uterine vein. Projecting into this space from the fetal side are the capillary rich chorionic villi, which allow for exchange between the two separate
Received: May 31 2013. Accepted: June 17 2013. Reprint request to: Dr Toshio Miki, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, 1425 San Pablo Street, BCC 412, MC 9080, Los Angeles, CA 90033, USA. Email: [email protected]
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circulations via simple and facilitated diffusion, active transport, and pinocytosis. The chorionic plate is the main body of the placenta, and houses the bulk of placental circulation. It receives blood from the fetus via the umbilical arteries and returns blood to the fetus via the umbilical vein. The membranes are composed of the amnion, the chorion and the decidua capsularis.3 The amnion consists of three layers with two types of cells. The inner most (fetal side) compact layer consists of amniotic epithelial cells attached to a basement membrane. The second layer reinforces the inner layer with mesenchymal stromal cells. The outer layer, called the spongy layer or zona spongiosa, is adjacent to the chorionic membrane and functions to protect the inner layers by absorbing mechanical stress from the outside. Both human amniotic epithelial cells (hAEC) and amniotic mesenchymal stromal cells (hAMSC) have been proposed to possess stem cell-like characteristics.4–6 In this review we provide a brief overview of the stem cell characteristics of these amnion-derived stem cells and focus on summarizing recent studies that have demonstrated therapeutic efficacy of these cells for liver diseases.
Unique Developmental Aspects of Amniotic Epithelial Cells The placenta consists of multiple layers derived from developmentally different origins. Unlike the bulk of the placenta, which is derived from trophectoderm, amniotic epithelial cells are derived from epiblasts. By the 8th day following fertilization, the inner cell mass (ICM) of the blastocyst differentiates into two layers, the hypoblast and the epiblast. The amniotic cavity develops within the epiblasts, and cells lining the cavity are termed amnioblasts, which eventually develop into amniotic epithelia (Fig. 1). The derivation of amnioblasts from epiblasts occurs about a week prior to gastrulation, which is the point at which the fate of most somatic cells is specified. The cells are further spatially segregated throughout gestation from the epicenter of organogenesis (embryo/fetus) by amniotic fluid. Therefore, it is speculated that the epiblast-derived amnioblasts do not receive differentiation signals in a systemic fashion. In spite of the rapid expansion of the amniotic membrane surface, the mitotic division rate of AEC is relatively low, peaking around day 20 (0.8%) and stopping by day 180.7
Figure 1 Developmental origin of amnion-derived stem cells. Schematic structure of early stage of human embryo (left) and hematoxylin–eosin staining of amnion in human term placenta (right) showing developmental origin of both human amniotic epithelial cells and amniotic mesenchymal stroma cells. The amnioblast (green) derived from pluripotent epiblasts at day 8 after fertilization. Concomitantly, extra-embryonic mesoblasts (red) arise and cover the interior of the cytotrophoblast and the exterior of the primary yolk sac as well as the amniotic cavity.
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The unique developmental environment of amniotic epithelial cells leads to the hypothesis that some amniotic epithelial cells may maintain epiblast-like characteristics throughout gestation until after the delivery.8 Indeed, embryonic stem cell surface markers are still expressed on a population of amniotic epithelial cells in term placentae.9,10 These include stage-specific embryonic antigen-3 (SSEA-3), tumor rejection antigen 1–60 (TRA1-60), and TRA1-81, which are expressed on 5–10% of hAEC. Furthermore, the number of stem cell marker positive cells is higher in the fetal amnion at 18–19 weeks (30–40%) than at term, indicating that the number of stem cells of epiblast origin stochastically decreases as pregnancy progresses.11 Taken together, these observations indicate that most of the AE cells spontaneously differentiate, and amniotic epithelium in term placenta contains cells in various stages of development. It is not yet clarified if this spontaneous differentiation is towards a specific single lineage or multiple lineages. Several groups have demonstrated the developmental potential of hAEC.9,12 Primary hAEC differentiate into cells of all three germ layers under appropriate culture conditions (Table 1). Although these data indicate that hAEC likely possess multipotency, this has not yet been proven at the single cell level. If the spontaneous differentiation of amnioblasts has occurred and this differentiation occurred randomly towards all three germ cell lineages, the primary hAEC population in term placenta contains all lineage-committed cells as well as stem cells. Each culture condition that has been used in order to induce in vitro differentiation might instead select particular lineage committed cells. Further studies must be conducted to clarify the pluri-/multipotency of a single hAEC. Table 1 Developmental potential of amnion-derived stem cells
AEC
AMSC
Lineage
Types of target cells
References
Ectoderm Mesoderm Endoderm
Neuron, glial cell Cardiomyocyte β-cells, hepatocyte
9,12
Ectoderm Mesoderm
Neuron Adipocyte, chondrocyte, osteocyte, skeletal myocyte, cardiomyocyte, endothelial cell β-cells, hepatocyte
19–21
Endoderm
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9,12,13 9,12,14–18
21–26
19,27
Origin of Amniotic Mesenchymal Stromal Cells When the amniotic cavity has developed, extraembryonic mesoblasts arise and line the interior of the cytotrophoblast, the exterior of the primary yolk sac, and the amniotic cavity. It is unclear if the extraembryonic mesoblasts are derived from the trophoblast or the epiblast.28 The extra-embryonic mesoblasts eventually develop into the amniotic connective tissue, including the AMSC (Fig. 1). As other mesenchymal stem cells, hAMSC are defined as plastic-adherent cells that can be subcultured several times, and display a specific cell surface antigen profile. Freshly isolated hAMSC express CD13, CD29, CD44, CD54, CD73, CD105, CD166 and the absence of CD34, CD45, CD14, and CD31. hAMSC demonstrate mesenchymal stem cell-specific differentiation potential into osteocytic, chondrocytic and adipocytic mesodermal lineages.19–25 In addition, studies have reported that hAMSC also possess differentiation potential toward neural (ectoderm),20,21 skeletal muscle, cardiomyocitic, endothelial (mesoderm),23,24 pancreatic, and hepatocytic (endoderm)19,27 lineages. In these reports, however, tested hAMSC were not carefully purified from the hAEC population. These non-mesodermal differentiation potentials might actually be provided by the contaminated lineage committed hAEC described above.
Immunomodulatory Properties of Amnion-derived Cells It is well recognized that amniocytes express low levels of MHC class I, which leads to low immunogenicity.29,30 The recent finding of expression of HLA-G and costimulatory molecules CD40L, CD80, CD86 and PD-1 also contributes to the observed immuneprivileged status of amnion-derived cells.13,26,31–34 These cells also produce and release Fas ligand, which further serves an immunomodulatory function. It has been reported by several groups that hAEC and hAMSC inhibit the allogeneic mixed lymphocyte reaction (MLR) in a dose-dependent manner35–37 as well as proliferation of T- and B-lymphocytes. Other studies have indicated that various soluble factors produced by amniocytes affect the T-cell response.38 Suggested potential humoral factors are tumor necrosis factor (TNF)-α Fas ligand (FasL), TNF-related apoptosisinducing ligand (TRAIL), transforming growth factor (TGF)-β,38 indoleamine 2,3-dioxygenase (IDO), and interleukin-10 (IL-10).39–41
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As the amnion layer is separated from the fetomaternal interface by the chorion, it is questionable if these unique immunomodulatory properties contribute to maintaining fetomaternal tolerance during pregnancy, and is instead due to the immaturity of these cells. Regardless of the role for these properties in nature, they provide a remarkable advantage for their use in cell therapy-based treatments.
Lack of Stem Cell Tumorigenicity Tumorigenicity of stem cells represents the key obstacle for safe clinical use of cell-based regenerative medicine therapies. Unlike ES cells, AE cells are not tumorigenic upon transplantation. In the early 1980s, human amnion was transplanted into patients in order to either test immunogenicity29 or to restore a glycogen deficiency.42–45 No tumor formation was reported in these clinical transplant cases. In addition to these clinical data, Miki et al. tested the tumorigenicity of hAEC using immunodeficient SCID/Beige mice.9 After observation throughout the lifespan of the recipient animals (∼2 years), no tumor formation was observed. This finding was subsequently confirmed by Ilancheran et al.12 This unique property may be due to the epigenetic state of these cells. Easley et al. conducted epigenetic profiling assays with the hAEC,46 and found that the global DNA methylation status of hAEC was intermediate between pluripotent stem cells (ES cells) and somatic cells (skin fibroblasts) while the histone acetylation status was similar to the somatic cells. This epigenetic modification status could explain the dichotomous property of hAEC that possess both stem cell-like plasticity (differentiation capability) and genetic stability (non-tumorigenic). These data clearly indicate the advantage for the potential clinical use of hAEC.
Hepatic Differentiation Potential of Amnion-derived Stem Cells The potential for hepatic differentiation of hAEC has been demonstrated in multiple studies. In the 1990s Sakuragawa’s group sought to take advantage of the low immunogenicity of hAEC for the purpose of utilizing these cells as gene carriers for the conduction of gene therapy.14 hAEC genetically labeled with the β-galactosidase gene were transplanted into the livers of SCID mice. Interestingly, the hAEC had integrated into the liver parenchyma and expressed both
α-fetoprotein and albumin at 1 and 2 weeks after cell injection. Takashima et al. conducted gene expression analysis on isolated hAEC and demonstrated that some, but not all hepatocyte marker genes were expressed following 2 weeks of culture in media supplemented with hepatocyte growth factor, fibroblast growth factor-2, and oncostatin M.15 Miki et al. identified embryonic stem cell marker positive cells in hAEC and demonstrated in vitro hepatic differentiation using a simple two-step protocol.9,16,17 Ilancheran et al. independently validated these results in 2007.12 In 2011, Marongiu et al. demonstrated that co-culture with mouse hepatocytes induces hepatic maturation in hAEC.18 Interestingly they also demonstrated that activin-A pretreatment, which is an essential step to induce endoderm specification in pluripotent stem cells, was not required for hepatic differentiation of hAEC. This result suggests that some of the hAEC were already committed to an endodermal lineage prior to the hepatic differentiation. As mentioned above, primary hAEC populations may contain both stem cell-like pluri/multi potent cells and endoderm lineage committed cells, with varying degrees of multipotency and lineage commitment. An efficient strategy for isolation of hepatic cells is to first induce in vitro hepatic differentiation in all primary AEC of a population, and afterwards sort out successfully derived hepatocyte-like cells for subsequent transplantation.
Strategy of Stem Cell Technology for Treating Liver Diseases End-stage liver failure is a devastating health-care problem worldwide. The World Health Organization estimates that approximately 10% of the world population, including 25 million Americans, has chronic liver disease. In the USA alone, over 40 000 patients progress to end-stage liver failure and death due to fulminant liver failure. The only effective treatment intervention at this stage is liver transplantation. However, fewer than 6000 liver transplants are performed annually in the USA, resulting in significant rates of waiting list morbidity and mortality. One option to bridge the gap between liver failure and liver transplant is hepatocyte transplantation.47,48 Hepatocyte transplantation (HTx) is a less invasive procedure than whole liver transplant, with less associated morbidity, fewer complications, a significantly reduced recovery time, and a lower cost. More than 50
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cases of clinical HTx have been conducted and have demonstrated therapeutic efficacy.49–53 This cell replacement approach is particularly suitable to treat congenital liver disorders as metabolic disease patients require only partial replacement of hepatocytes to compensate for the particular missing enzyme function.54 In a patient with Crigler–Najjar syndrome, an infusion of normal hepatocytes, equivalent to 5% of the parenchymal mass, was able to achieve a medium-term reduction in serum bilirubin.55 Similarly, a child effected by ornithine transcarbamylase deficiency who received 1.9x109 hepatocytes demonstrated normalization of plasma ammonia and glutamine levels on a normal diet without phenylbutyrate/phenylacetate therapy.56 These cases clearly demonstrate that congenital metabolic disorders can be effectively treated via cell replacement therapy. Currently, the shortage of donor hepatocytes prohibits extensive clinical application of HTx, making stem cell-derived hepatocytes an attractive alternative source.57 Embryonic stem cells and induced pluripotent stem cells are promising sources for the production of hepatic cells, and numerous studies have demonstrated the hepatic differentiation potential of pluripotent stem cells in vitro. The intrinsic risk of teratoma formation and the genetically unstable nature of these cells prevent clinical applications.58 As described above, hAEC possess hepatic differentiation ability and do not form tumors upon transplantation, making them perhaps the most clinically applicable stem cell-derived hepatic cells for this purpose.
Congenital Liver Metabolic Disorders Approximately 1 in 1500 children is born with a congenital metabolic disease (CMD).59 Many of these critical inborn errors of metabolic or synthetic processes principally involve the liver. Current therapy for CMD consists of life-long dietary restrictions with or without supplementation of amino acids. Several preclinical studies using disease model mice provide promising evidence regarding the therapeutic potential of hAEC transplantation and will be summarized below.
Maple Syrup Urine Disease Maple syrup urine disease (MSUD) is a genetic disorder caused by a deficiency of branched-chain alphaketo acid dehydrogenase complex due to mutations in the BCKDHA, BCKDHB, DBT, or DLD genes.60 These
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mutations impair the oxidative decarboxylation of branched-chain keto acids derived from branchedchain amino acids (BCAA). As a result, BCAA, leucine, isoleucine and valine as well as their byproducts accumulate and lead to severe organ damage. The current treatment, lifelong dietary restriction of protein and BCAA, has yielded largely unsatisfactory results, suggesting that new approaches to treat this disease are required. In 2006, Homanics et al. produced an intermediate MSUD (iMSUD) disease model mouse with relevant similarities to the human disease.61 Unlike classic MSUD model mice, disease onset is delayed in the iMSUD mice.62 Skvorak et al. transplanted primary hAEC directly into the liver of neonatal iMSUD mice,63 which significantly improved survival of recipient animals at 35 days (100%) and 100 days (81.2% survival). All untreated control animals died before 30 days of age. The bodyweight of hAEC-recipient mice was normalized along with the restoration of general health. BCAA levels were also significantly improved in the serum and in the brain. Although engrafted hAEC in the recipient liver were not present in the observed histologic sections, expression levels of human BCKDHA, BCKDHB, and DLD mRNA in the liver at 100 days indicate hAEC had engrafted and were enzymatically active in the recipient mouse liver for more than 3 months. This preclinical study demonstrated the remarkable potential of hAEC transplantation. Further studies with clinically applicable grade hAEC are eagerly anticipated to lead to clinical trials.
Niemann–Pick Disease Niemann–Pick disease (Type C1) is also a genetic disorder caused by a mutation of a gene (NPC1) that contributes to intracellular transport of cholesterol and lipids.64 This resulting sphingomyelinase deficiency leads to an accumulation of excess intracellular lipids and leads to both severe liver damage and neurodegeneration. As the pathological condition is systemic, the therapeutic concept of hepatocyte transplantation at first glance does not appear suitable for the treatment of this disease. Hong et al., however, utilized hAEC as a source of enzyme replacement rather than cell replacement in a mouse model of Niemann– Pick disease.65 They injected half a million hAEC every other week from 5 weeks of age. This treatment significantly extended the lifespan of the test group, and resulted in alleviation of tissue damage. The same concept was tested in 1987 by the implantation of human amnion (membrane) into a subcutaneous
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thoracic pouch under the armpit of a 14-year-old Niemann–Pick disease type B patient.42 A total of six implantations were carried out at 1–4-month intervals. Immediately after the first implantation, clinical improvements were observed. Serum linoleic acid level was also significantly increased within 6 months. The therapy was stopped when the subcutaneous pouch could no longer tolerate further implantations. In 1992, Bembi et al. reported on five type B patients in which hAEC were implanted in order to supply exogenous sphingomyelinase.66 After 4 months, the sphingomyelinase activity achieved levels seen in heterozygotes. In two cases, a single implantation normalized urinal sphingomyelin and phospholipid levels. If longterm survival of hAEC can be achieved, these promising results suggest the potential of future therapy for Niemann–Pick disease utilizing hAEC.
Liver Cirrhosis Liver cirrhosis is the end-point of liver fibrosis in many chronic liver conditions, including viral infections, alcohol abuse, drug/chemical administrations, and exposure to radiotherapy. Cirrhosis and chronic liver disease were the 10th leading causes of death for men and the 12th for women in the USA in 2001.67 Cirrhosis occurs when scar tissue replaces the normal liver parenchyma, which impairs function, and blocks normal blood flow through the organ. The cirrhotic liver is not a suitable environment for cell transplantation, regardless of which cell type is used. If cell replacement therapy is conducted before end-stage cirrhosis, however, cell therapy may delay or prevent disease progression. Indeed, some preclinical studies indicate that amnion-derived cell transplantation improves liver fibrosis in animal models. Manuelpillai et al. systemically (via tail vein) transplanted hAEC to a carbon tetrachloride (CCL4)-treated liver cirrhosis mouse model.68 The biochemical marker of liver damage (alanine aminotransferase) significantly improved and hepatocyte apoptosis was reduced after 2 weeks of hAEC transplantation. The study also demonstrated the reduction of hepatic stellate cell activation and hepatic fibrosis. Although direct evidence was not presented, it suggested an increased matrix metalloproteinase (MMP)-2 activity as the mechanism underlying the fibrosis regression. Zhang et al. transplanted human amnion-derived cells into CCL4treated mouse liver via the spleen.69 They also demonstrated an improved ALT score, and a decrease of both apoptosis and fibrosis. Although the amnion-
derived cells were originally hAEC, it appeared after in vitro expansion that the mesenchymal cell population was the dominant cell population. The mechanism of this anti-fibrosis effect should therefore not be considered a property of hAEC, rather more likely a general mesenchymal stem cell property. Manuelpillai followed up the previous study and proposed a novel anti-fibrosis mechanism of hAEC transplantation.70 They demonstrated that transplanted hAEC activate M2 (healing) macrophages rather than M1 (proinflammatory)-type macrophages. Anti-fibrotic effects of amnion-derived cells have also been observed by covering the liver surface with human amniotic membrane. Ricci et al. patched fresh or cryopreserved human amnion membrane onto the surface of rat livers with induced fibrosis, and evaluated the number of α-smooth muscle actin, Desmin, and CK19-positive cells in the treated liver.71 The number of fibroblast marker positive cells was decreased in the amnion-patched group, on both the surface and in the core of the liver. In the follow-up studies conducted by the same group, the researchers concluded that prostaglandins are the key effectors of the immunomodulatory activity of amniotic membrane.72 In summary, transplanted hAEC appear to function in the treatment of cirrhosis by multiple routes, including: secretion of MMP that induce fibrinolysis, inactivation of hepatic stellate cells by a decrease in TGFβ-1 and an increase in IL-10 concentration, and activation of M2 type macrophages. These studies have provided important data; however, they did not conclusively demonstrate these pathways and the interactions of each mechanism. They also did not elucidate the crosstalk between the engrafted hAEC and the recipient’s immune cells. More detailed studies are needed to explain these fundamental questions.
Conclusion In this review we have briefly introduced the hypotheses explaining why AEC, like stem cells, demonstrate developmental potential to all three germ layers.8 We have also focused on potential clinical applications of amnion-derived cells for the treatment of liver diseases, an area in need of novel therapeutic approaches. As summarized here, preclinical studies with disease model animals have successfully demonstrated the therapeutic potential of hAEC transplantation. However, there are still many issues that must be clarified before these cells can be used for the treatment of
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patients. Clinically applicable cells must demonstrate scalability, reproducibility, and purity. Further studies are essential to identify the cell surface markers required for isolation of a pure population, as well as culture conditions that enable expansion of cell numbers without altering cell characteristics. The immunomodulatory effects of hAEC must be comprehensively characterized both in the immediate period following transplantation, and long-term. Amnionderived stem cells possess advantages due to their unique developmental origin. The amnion-derived cells are readily available, pluripotent/multipotent cells that are generated from an ethically acceptable tissue. We must continue accumulating scientific evidence to safely utilize the therapeutic potential of hAEC.
Acknowledgments T.M. is supported by California Institute for Regenerative Medicine (CIRM) grant TR3-05488.
Disclosure The authors have no financial support or relationship that may pose conflict of interest.
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