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Human amniotic fluid: a source of stem cells for possible therapeutic use Margaret Dziadosz, MD; Ross S. Basch, MD; Bruce K. Young, MD

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tem cells have become the focus of great interest because of their potential for therapy in a wide variety of conditions. Conditions possibly treatable by stem-cell transplants include genetic defects, tissue and organ replacement, autoimmune disease, and malignancies. A stem cell is an undifferentiated cell that is capable of prolonged self-replication without differentiation. There is a spectrum of stem cells that ranges from multipotent cells that have the potential to differentiate into 1 or 2 lineages, pluripotent cells that may differentiate into many lineages, and totipotent cells that have unlimited differentiation potential. A totipotent stem cell should have the potential to differentiate into lineages for mesodermal, ectodermal and endodermal tissues (such as osteogenic, neurogenic, and hepatic lineages).1 Stem cells are characterized by the presence of surface markers and transcription factors that are associated with self-renewal without differentiation as seen in early embryonic states. Some examples of these surface markers are From the Division of Maternal Fetal Medicine, Department of Obstetrics and Gynecology (Drs Dziadosz and Young) and the Department of Pathology (Dr Basch), New York University Langone Medical Center, New York, NY. Received Sept. 1, 2015; revised Oct. 22, 2015; accepted Dec. 31, 2015. There was financial support from The William and Linda Haugland Foundation for laboratory research cited, but they had no involvement in study design, data collection, data analysis, interpretation, or writing of this report or in the decision to submit this manuscript for publication. The authors report no conflict of interest. Corresponding author: Bruce K. Young, MD. [email protected] 0002-9378/$36.00 ª 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajog.2015.12.061

Stem cells are undifferentiated cells with the capacity for differentiation. Amniotic fluid cells have emerged only recently as a possible source of stem cells for clinical purposes. There are no ethical or sampling constraints for the use of amniocentesis as a standard clinical procedure for obtaining an abundant supply of amniotic fluid cells. Amniotic fluid cells of human origin proliferate rapidly and are multipotent with the potential for expansion in vitro to multiple cell lines. Tissue engineering technologies that use amniotic fluid cells are being explored. Amniotic fluid cells may be of clinical benefit for fetal therapies, degenerative disease, and regenerative medicine applications. We present a comprehensive review of the evolution of human amniotic fluid cells as a possible modality for therapeutic use. Key words: amniotic fluid, stem cell, therapy, transplantation

SSEA3, SSEA4, Tra-1-60, Tra 1-81, CD117, and CD90. Some important transcription factors that are associated with embryos are Oct3, Oct4, Sox2, Nanog, and Rex1. Flow cytometry has been the primary technique for the identification of stem cells by detection of these markers. Stem-cell therapy introduces stem cells into selected tissue environments to prevent and treat injury or repair abnormal tissue. Stem cells from various sources have been considered as potential therapeutic alternatives to organ replacement and other therapies that require transplantation. They can be studied in vitro by modeling pathophysiologic processes of inflammation, disease, and treatments that would occur in vivo. They are introduced intravenously or directly into tissue to study the prevention of injury or the repair of a damaged system or congenital defect. Stem cells are well-known to exist in embryonic tissue and bone marrow. Additional sources of stem cells in humans have been identified: umbilical cord blood, umbilical cord cells, placenta, amniotic membranes, amniotic fluid, peripheral blood, and somatic cells induced to pluripotency. For induced pluripotent cells that are derived from human somatic cells, Oct4, SOX2,

KlF4, and c-myc also have been used to transduce somatic cells to develop a pluripotent cell population. There has been significant controversy regarding the various sources about safety in transplantation, accessibility, and ethical issues when human embryos are the source, tumorigenesis with embryonic and transduced cells, and expansion and reliability of all these sources for clinical use.2 Therefore, amniotic fluidederived cells might be a practical alternative source because they are readily available, grow well in culture, and avoid these issues.

Alternative sources of stem cells Embryonic stem (ES) cells are totipotent but have been shown to form tumors in immunodeficient mice. ES cells grow as teratocarcinomas in vivo and frequently acquire chromosomal aberrations.3,4 They have restricted differentiation capacity and have been shown to yield both genetic and epigenetic abnormalities in culture. Significant ethical issues arise regarding the use of embryos as well. Cord blood cells are expanded easily but are primarily hematopoietic lineage cells with <1% multipotent cells. There is substantial clinical experience with cord blood transplantation for

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hematologic indications and growing interest in other applications.5,6 Umbilical cord and placental stem cells exist in the middle ground between ES cells and adult stem cells and might yield desirable expansion with lack of tumorigenesis. However, expansion and culture studies are limited at present. Bone marrow is the most common source of adult stem cells for clinical transplantation. Restricted lineage, low proliferation rates because of limited telomerase expression, and restricted differentiation potential limit their clinical application so far. They are, however, less tumorigenic than ES cells and do not have ethical concerns. There is extensive clinical experience with their use, establishing standard protocols for transplantation. Amniotic fluid is readily available, is noncontroversial, and is obtained routinely by amniocentesis. It is analyzed for prenatal genetic diagnosis in the second trimester, for evidence of fetal pulmonary maturity in the third trimester, and for infection at any gestational age. It is made up of a heterogeneous population of cells that routinely is retrieved clinically and cultured for genetic studies. It has been shown that a significant percentage of cells that are obtained from amniocentesis exhibit stem-cell markers. These cells are cultured easily, expanded, and remain viable over many passages, tolerating cryopreservation very well.2,7-10 A great deal of research has been performed on second-trimester amniotic fluid cells (AFC), but there is little known about whether third-trimester AFC closely resemble those of the midtrimester.11 You et al11 retrieved amniotic fluid at the time of elective cesarean delivery at term. AFC were isolated in culture and found to be positive for surface markers CD29, CD73, CD90, and CD105 as well as Oct-4. Proliferation potential was verified, and differentiation into osteogenic lineage was reported as successful. A more recent study aimed to characterize human AFC from the early third trimester, where 3 samples were analyzed from gestations from 28-34 weeks. Third-trimester AFC expressed comparable levels of Oct-4 and Nanog, but lower levels of SOX2

ajog.org and Rex-1. Samples were also successfully differentiated to adipocytes, osteoblasts, chondroblasts, myocytes, and neural-like cells, although thirdtrimester samples showed poor differentiation potential to myocytes and stronger potentiation to neural lineage.12 Further studies on not only thirdtrimester amniotic fluid but also AFC at term are warranted, because these findings are promising but inconclusive.

AFC In early studies, amniotic fluid from pregnant ewes was isolated and expanded to mesenchymal, fibroblast/myofibroblast cell lineages. These cells were noted to proliferate significantly faster than surrounding cells, showed little cell death, and could be isolated consistently from amniotic fluid.13 AFC showed stem-cell potential when they were found to contain Oct-4, which is a known marker specific for human embryonic stem cells that are associated with maintenance of the undifferentiated state and pluripotency.14,15 Besides their rapid proliferation, human AFC have been differentiated successfully into all embryonic germ layers, thereby demonstrating pluripotency. After multiple passages in culture, human AFC remained chromosomally stable and did not form teratomas or undergo malignant change.2,7 These qualities of human AFC suggested significant advantages as a potential source of cells for clinical transplantation. ES cell molecular markers are molecules that specifically are expressed by stem cells and are critical to the characterization and identification of their pluripotency. There are a wide range of cell-surface proteins, transcription factors, and molecular markers indicative of stemness (Table). These surface markers are usually glycosphingolipids or membrane proteins. Research on stem cells has used the technique of flow cytometry. Flow cytometry separates cells using their cell surface markers, thereby identifying viable cells. This process then permits the formation of clonal cultures from specifically identified cells. Flow cytometry can also be used to identify transcription factors in

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cell nuclei that are related to stem-cell behavior. However, in contrast to the process of identification of cell surface markers, the cells are no longer viable and cannot be cultured after transcription factor identification. Human amniotic fluidederived stem cells were identified as expressing Oct-4, SOX2, Nanog, Rex1, cyclin A, and mesenchymal markers that include CD90, CD105, CD73, CD166, CD133, and CD44.15-19 There is a higher percentage of stem-cell transcription factors Oct-4, Nanog, and SOX2 in AFC from fluid that is obtained from 15-17 weeks gestation vs later gestational ages. Cell surface markers do not appear to vary with gestational age among the second-trimester samples, but individual samples’ expression varies greatly and may overshadow this effect.10 The ability for adipocyte, osteocyte, and neuronal cell generation were also demonstrated.20,21 De Coppi et al21 postulated that CD117 was a marker for selection of AFC from human amniocentesis cultures. However, subsequently, it was shown that CD117 is actually a clonal marker that was present in only approximately 0.5-2% of AFC in cultures that used the same media.10,22 Another variety of human AFC, mesenchymal stem cells have been identified with distinct populations of varying differentiation potential.23 Our studies on human AFC have shown the presence of CD117, 133, 90, 15, 44, 29, 9, 73, as well as SSEA1, SSEA3, SSEA4, Tra-1-60, Tra-1-81, Oct4, Rex1, Nanog, and SOX2.24-28 Our laboratory selected SSEA4, Tra-1-60, and CD90 for subsequent investigations because they were the most highly expressed ES cell markers in our patient samples. These studies have also shown clonal populations bearing all 3 markers, combinations of 2 different stem cell markers (eg, SSEA4/CD90, SSEA4/TRA-1-60, and Tra-1-60/CD90), and populations with just 1 marker.10 Clones with different combinations of markers may vary in their properties of stemness. This is an area for further investigation that has not yet been explored. Thus, there is a mixture of cells with varying potential

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TABLE

Human embryonic stem-cell markers24-28 Marker

Characteristic

Classification

Known activity

SSEA-1 (CD15/Lewis x)

EC, ES

Carbohydrate-associated; lactoseries oligosaccharide antigen

Increase with differentiation

SSEA-3 (glycolipid GB5)

EC, ES, EG

Carbohydrate-associated; globoseries carbohydrate antigen

Decrease with differentiation

SSEA-4

EC, ES, EG

Carbohydrate-associated glycoprotein

Decrease with differentiation

Nanog

ES

Homeobox family, DNA-binding transcription factor

Decrease with differentiation

Oct-4 (POU5F1)

EC, ES, EG

Octamer-binding transcription factor

Most recognized marker totipotent ES

Rex-1 (Zfp42)

EC, ES

Zinc finger family

Maker of undifferentiated cells

SOX2

EC, ES

SOX family, DNA-binding transcription factor

Specifies germ lineage at implantation, regulates proliferation, differentiation

TRA-1-60

EC, ES, EG

Surface antigen, epitope expressed on high molecular weight proteoglycan

Tumor recombinant antigen

TRA-1-81

EC, ES, EG

Surface antigen, epitope expressed on podocalyxin

Tumor recombinant antigen

CD9 (MRP1)

ES

Surface marker, tetraspanin family

Cell adhesion, migration, T-cell stimulation

CD29 (B1 integrin)

ES

Surface marker, intregrin family

Cell adhesion, recognition, embryogenesis

CD44

EC, ES

Surface marker, glycoprotein

Cell interactions, adhesion, migration

CD45 (PTPRC)

ES

Protein tyrosine phosphatase receptor; leukocyte common antigen

Cell growth and differentiation, mesenchymal, hematopoetic cells

CD73

ES

Surface marker, enzyme

Converts adenosine monophosphate to adenosine

CD90 (Thy-1)

EC, ES

Surface marker, GPI-linked glycoprotein

Mesenchymal stromal cells, hematopoietic stem cell, neuron, T-cell activation

CD105

ES

Surface marker, endoglin, transmembrane glycoprotein of zona pellucida

Differentiation of smooth muscle, angiogenesis, neovascularization

CD117 (c-kit)

EC, ES

Surface marker, stem-cell factor receptor, tyrosine kinase receptor

Present on hematopoietic progenitor cells, role in gametogenesis

CD133

EC, ES

Surface marker, Prominin-1, glycoprotein

Organizer of cell membrane topology

CD166

EC, ES

Surface marker, immunoglobulin

Cell interaction and adhesion

Stage-specific embryonic antigens (cell surface associated)

Transcription factors

Cluster of differentiation markers

EC, embryonic carcinoma cells; ES, embryonic stem cells; EG, embryonic germ cells; GPI, glycosylphosphatidylinositol. Dziadosz. Amniotic fluid for possible therapy. Am J Obstet Gynecol 2016.

for differentiation into lineages from all 3 germ layers, likely in addition to cells that are pluripotent. As yet, it is not clear which clones will have different potentials for differentiation, but clonal isolation of a single

pluripotent clonal cell line is not essential for AFC differentiation. Because of the heterogeneous nature of AFC, it may not be important to isolate a pluripotent stem-cell clone to build a framework with which to move towards

translational use in therapy.10 Our laboratory studied 37 different samples of human amniotic fluid. A total of 81 cultures were obtained for study from doubling each culture sample from 2-8 times. With every culture, there was

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consistent inducibility to osteogenic, chondrogenic, and neurogenic lineage without the need for isolation of a specific clone with either a single marker or combination of markers.10 Individual variation in the percentage distribution of cells that express different markers does not appear to be a limitation because every sample has a wide variety of lineage precursors. Amniotic fluide derived cells have been reprogrammed easily into induced pluripotent stem cells, which supports this concept.21 This is in contrast to the concerns raised by Ekblad et al.29 They focused on selecting a single CD117-positive pluripotent cell as proposed by Zia et al22 and were unsuccessful in enriching the CD117 population. Thus, it is likely that clonal selection is unnecessary for clinical use.

Translational properties Amniotic fluid is retrieved from the amniotic sac at amniocentesis. It is common to use 3-5ecc aliquots of amniotic fluid. In our laboratory, this small amount yields an average of 100,000 cells per cubic centimeter with 60-90% viability. Once AFC are isolated from the amniotic fluid, they are passaged and doubled in an average of 36 hours. Therefore, in 72 hours or 2 passages, approximately 1.6 million cells become available. Of this total number, approximately 75% are viable or 1.2 million and up to 99% exhibit stem-cell markers.10 This is more than sufficient for laboratory studies. For clinical purposes, at least 5 cc of amniotic fluid may be necessary, depending on the number of cells that are required for transplantation and the time window for a given treatment. This will have to be determined in future clinical trials. Culture characteristics of AFC include longevity. Our laboratory has shown proliferation capacity of up to 20 passages, which can expand a cell population from a single sample aliquot to up to >10 billion cells.10 Cell lines have been propagated more than 250 times with retention of telomere length and a normal karyotype.21 Their cryopreservability and inducibility to multiple lineages have been reported by different

ajog.org groups.7,10 This differentiation into multiple fates has been documented via messenger RNA lineage specific genes and by histochemistry and histology.19 AFC have been reported to have a low immunogenic profile with minimal expression of HLA-ABC and no HLADR antigens.10,23 This could reduce the risk of rejection and of graft-vs-host disease. However, our recent work in collaboration with Carriere et al (unpublished data) has demonstrated the presence of HLA-DR antigens with the use of DNA technology in contrast to previous reports. This raises the question of the need for HLA matching with other than autologous transplantation. Thus, it is possible that some immunologic matching would be recommended for clinical transplantation.

Preclinical models The use of preclinical models has been promising for future therapeutic possibilities. Following reconstructive engineering concepts that were pioneered by Fauza et al30,31 in 1998, when gravid ewes’ fetal tissue was harvested and manipulated for autologous transplantation for injured fetal bladder, skin, and diaphragm, Kaviani et al13 engineered models with amniotic fluid cells. Amniotic fluid was obtained from pregnant ewes and expanded into mesenchymal, fibroblast, and myofibroblast cell lineages. Amniotic fluid mesenchymal stem cells transplanted to fetal lambs with intentional skin wounds yielded improved and more rapid healing time.33 Using a mouse model, Sun et al34 differentiated human AFC into keratinocytes that improved epidermal regeneration at intentional excisional wounds. Mesenchymal amniocytes that were grown on seeded scaffolds inserted to sites of diaphragmatic defects in fetal lambs showed improved structural repair over myocyte or acellular grafts.32 Recently, intraamniotic delivery of neurally induced amniotic fluid cells for fetal lambs with spina bifida was performed. Clustering of the induced cells in the neural placodes was seen, supporting regenerative therapeutic potential for transplanted AFC for the treatment of neural tube defects.36

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AFC were used to engineer autologous fetal tissue for body-wall defects, myelomeningocele, bladder extrophy defects, and congenital diaphragmatic hernia, also shown in a mouse model.35,37 Intratracheal injection of human AFC into a rabbit model of congenital diaphragmatic hernia also showed promising effects on improved lung density and function.38 Amniotic fluid cells have been shown to repair injured urinary bladder detrusor muscle and increase muscle strength in mice that had been modeled for spinal muscular atrophy.7,39 In mice with acute tubular necrosis caused by either rhabdomyolysis or cisplatin toxicity, infusion of AFC improved renal function as AFC migrate to areas of ischemia.40,41 AFC with renal progenitor phenotype have demonstrated a nephroprotective effect in an acute ischemia reperfusion model using rats that resulted in reduced creatinine level, less tubular necrosis, and less long-term fibrosis.42 Scaffolds provide an improved environment for cell growth and organogenesis. They are also a fundamental component for development of multidimensional structures. AFC are able to form tissue-engineered bone from printed scaffold constructs of osteogenically induced lineage cells in mice.13,21 The ability to create 3-dimensional bone constructs in rabbits shows promise for craniofacial reconstruction.43,44 There has been evidence of inducibility to cardiogenic lineage forming heart syncytia that pulsate, and vascular grafts have been produced by seeding 3-dimensional scaffolds.45,46 AFC systemic transplantation has also been shown to be cardioprotective and proangiogenic in a rat model of induced cardiac ischemia.47 AFC induced to neural lineage and exposed to nerve growth factor have been shown to facilitate peripheral nerve regeneration after sciatic nerve crush injury in rats.48 Ischemic stroke mouse and rat models have also been used to study the benefit of AFC therapies.49 No deformation of natural anatomy or malignant process developed in treated rat brains, and there was improvement in not only cognitive ability but also in

ajog.org rat behavior after treatment.50 In mice, intracerebroventricular administration of AFC also significantly reduced neurologic sequelae and behavior deficits after cerebral ischemia was induced via middle cerebral artery occlusion.51 Animal models with human AFC have been used to study neurodegenerative diseases of demyelination and neuronal loss mimicking Parkinson’s disease. The cells wholly integrate into mice brains, becoming indiscernible from surrounding tissue. The transplanted stem cells migrated towards areas of damaged neural tissue and resulted in symptomatic improvement.21 Endodermic lineages have also had promising results through AFC therapy. Injured liver in mice that were treated with AFC have shown the production of mature hepatocytes and progenitors that appear therapeutic.52 AFC have been transplanted to animal models with beneficial effects on necrotizing enterocolitis, which is a severe gastrointestinal disease that is common in premature neonates.53 Although AFC have not been differentiated successfully into lung tissue to date, the cells engraft and express alveolar and bronchial markers in the background of lung injury.54 Human AFC have been induced easily to pluripotency for possible autologous or homologous matched transplantation.55,56 Our laboratory has cultured human AFC in a serum-free medium, which is a prerequisite for clinical use and transplantation. Comparison of serum-free cultured cells with the same patient’s cells grown in standard cultures showed retention of stem-cell markers, similar proliferation potential, and no difference in the ability to be differentiated after cryopreservation into neural, osteoid, and cartilage cell lineages.9 It seems likely that, even without a cell line induced to pluripotency, AFC could be used for transplantation. Although in just the beginning phases, human transplantation with fetal stem cells has been promising. Fetal neural tissue from human fetal ventral mesencephalic tissue has been reported as a restorative treatment for patients with Parkinson’s disease with evidence of

Obstetrics prolonged symptomatic relief.57 Umbilical cord blood nucleated cells can differentiate into cells of neural lineage.58,59 In another study, fetal autologous umbilical cord blood has been transplanted into infants with neonatal hypoxic ischemic encephalopathy via the intravenous route. The study showed that autologous transplantation of fetal cells is feasible and that further clinical trials should be pursued.5 Fetal allogeneic HLA-mismatched mesenchymal stem cells have been transplanted to a fetus with in utero diagnosis of severe osteogenesis imperfecta. No immune reaction was detected against transplanted cells at 9 months of life, and the child has exhibited significantly decreased numbers of fractures from the expected dismal prognosis of severe osteogenesis imperfecta.60 A recent study in mice has demonstrated immunoregulatory properties of human AFC transplants that resulted in immunosuppression of T cells in regional lymph nodes.61 There are limited clinical trials currently ongoing that involve human transplantation of human AFC for therapeutic interventions. NuCell (Nutech Medical Inc., Birmingham, AL) is experimenting with an amniotic allograft, ReNu, that is composed of particularized amniotic membrane and cells from amniotic fluid and currently is recruiting patients in a multicenter study. The primary outcome involves improvement of pain scores after direct site injection for people with osteoarthritis-causing knee pain (Identifier NCT02318511).

Future developments AFC collected at routine amniocentesis could be banked and proliferated in culture, as needed.62 AFC banking would be a convenient source for autologous therapies. Even if HLA matching should be required, AFC could be used for therapy in histocompatible HLA typematched recipients and possibly used for fetal therapy. We are continuing to investigate the HLA immunogenicity of AFC to address this potentiality. In the evolving field of regenerative medicine that aims to replace or regenerate cells,

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tissue, or organs to restore normal function, AFC may be used to create grafts for reconstructive therapy. There are ongoing studies that are using biocompatible lattices and other stemcell sources that would be readily adaptable for AFC.63 They could be transplanted in the prenatal or postnatal period for diseases such as periventricular leukomalacia and neonatal encephalopathy and traumatic brain injury. Studies that are using cord blood are already underway, and similar studies with the use of AFC are feasible today.5,6 The establishment of a bank would be ideal for possible therapy in such a wide range of indications. The challenges will be to obtain Food and Drug Administration approval to initiate clinical trials, although basic preclinical work in humans will continue. Among these, the identification of a single pluripotent cell and cloning of that population remains to be accomplished.

Comment There is enormous potential in the growing field of regenerative medicine, clinical transplantation, and therapies that target morbidities that are associated with dysgenetics and birth injury.56 AFC can be retrieved easily during routine amniocentesis with minimal risk to the mother and fetus. Advantages include a high yield of cells per sample retrieved, a high proliferation rate, a differentiation potential into all embryonic germ cell layers, and the stability of both genetics and phenotype. In fact, this has been shown after 250 populations without change in karyotype. AFC also have slow onset of senescence and maintain normal telomere length.10 AFC show no evidence of tumor formation, including late passage cells.21,23 Low tumorigenic risk is essential for therapeutic use. Disadvantages include the small risk of the amniocentesis procedure including an estimated 0.1% risk of pregnancy loss in mid trimester. In the future, it may be ideal to collect amniotic fluid for AFC at term. Human AFC are heterogeneous with diversity among donors,29 but all amniotic fluids have large numbers of cells with multipotency. Further clinical trials must be

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explored, but clinical use remains a promising possibility. REFERENCES 1. Siegel N, Rosner M, Hanneder M, Freilinger A, Hengstschlager M. Human amniotic fluid stem cells: a new perspective. Amino acids 2008;35: 291-3. 2. Da Sacco S, De Filippo RE, Perin L. Amniotic fluid as a source of pluripotent and multipotent stem cells for organ regeneration. Curr Opin Organ Transpl 2011;16:101-5. 3. Cowan CA, Klimanskaya I, McMahon J, et al. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med 2004;350: 1353-6. 4. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981;292:154-6. 5. Cotten CM, Murtha AP, Goldberg RN, et al. Feasibility of autologous cord blood cells for infants with hypoxic-ischemic encephalopathy. J Pediatr 2014;164:973-9.e1. 6. Sun JM, Kurtzberg J. Cord blood for brain injury. Cytotherapy 2015;17:775-85. 7. De Coppi P, Callegari A, Chiavegato A, et al. Amniotic fluid and bone marrow derived mesenchymal stem cells can be converted to smooth muscle cells in the cryo-injured rat bladder and prevent compensatory hypertrophy of surviving smooth muscle cells. J urol 2007;177:369-76. 8. Da Sacco S, Sedrakyan S, Boldrin F, et al. Human amniotic fluid as a potential new source of organ specific precursor cells for future regenerative medicine applications. J urol 2010;183:1193-200. 9. Young BK, Chan MK, Liu L, Basch RS. Amniotic fluid as a source of multipotent cells for clinical use. J Perinat Med 2015. Epub ahead of print. 10. Chen Z, Chan MK, Steichenko N, et al. Heterogeneity of stem cells in human amniotic fluid. J Regen Med 2014;3:1-8. 11. You Q, Tong X, Guan Y, et al. The biological characteristics of human third trimester amniotic fluid stem cells. J Int Med Res 2009;37:105-12. 12. Savickiene J, Treigyte G, Baronaite S, et al. Human amniotic fluid mesenchymal stem cells from second- and third-trimester amniocentesis: differentiation potential, molecular signature, and proteome analysis. Stem Cells Int 2015;2015:319238. 13. Kaviani A, Perry TE, Dzakovic A, Jennings RW, Ziegler MM, Fauza DO. The amniotic fluid as a source of cells for fetal tissue engineering. J Pediatr Surg 2001;36: 1662-5. 14. Pan GJ, Chang ZY, Scholer HR, Pei D. Stem cell pluripotency and transcription factor Oct4. Cell res 2002;12:321-9. 15. Pesce M, Scholer HR. Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells 2001;19:271-8.

ajog.org 16. Prusa AR, Marton E, Rosner M, Bernaschek G, Hengstschlager M. Oct-4e expressing cells in human amniotic fluid: a new source for stem cell research? Hum reprod (Oxford) 2003;18:1489-93. 17. In ‘t Anker PS, Scherjon SA, Kleijburg-van der Keur C, et al. Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation. Blood 2003;102:1548-9. 18. Karlmark KR, Freilinger A, Marton E, Rosner M, Lubec G, Hengstschlager M. Activation of ectopic Oct-4 and Rex-1 promoters in human amniotic fluid cells. Int J Mol Med 2005;16:987-92. 19. Bossolasco P, Montemurro T, Cova L, et al. Molecular and phenotypic characterization of human amniotic fluid cells and their differentiation potential. Cell Res 2006;16: 329-36. 20. Prusa AR, Marton E, Rosner M, et al. Neurogenic cells in human amniotic fluid. Am J Obstet Gynecol 2004;191:309-14. 21. De Coppi P, Bartsch G Jr, Siddiqui MM, et al. Isolation of amniotic stem cell lines with potential for therapy. Nature biotechnol 2007;25:100-6. 22. Zia S, Toelen J, Mori da Cunha M, Dekoninck P, de Coppi P, Deprest J. Routine clonal expansion of mesenchymal stem cells derived from amniotic fluid for perinatal applications. Prenat Diagn 2013;33:921-8. 23. Trohatou O, Anagnou NP, Roubelakis MG. Human amniotic fluid stem cells as an attractive tool for clinical applications. Curr Stem Cell Res Ther 2013;8:125-32. 24. Young RA. Control of the embryonic stem cell state. Cell 2011;144:940-54. 25. Chambers I, Colby D, Robertson M, et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003;113:643-55. 26. Kaplan R, Morse B, Huebner K, et al. Cloning of three human tyrosine phosphatases reveals a multigene family of receptor-linked protein-tyrosine-phosphatases expressed in brain. Proc Nat Acad Sci USA 1990;87:7000-4. 27. Bowen MA, Patel DD, Li X, et al. Cloning, mapping, and characterization of activated leukocyte-cell adhesion molecule (ALCAM), a CD6 ligand. J Exper Med 1995;181:2213-20. 28. Zhao W, Ji X, Zhang F, Li L, Ma L. Embryonic stem cell markers. Molecules 2012;17: 6196-236. 29. Ekblad A, Qian H, Westgren M, Le Blanc K, Fossum M, Gotherstrom C. Amniotic fluid-a source for clinical therapeutics in the newborn? Stem Cell Dev 2015;24:1405-14. 30. Fauza DO, Fishman SJ, Mehegan K, Atala A. Videofetoscopically assisted fetal tissue engineering: bladder augmentation. J Pediatr Surg 1998;33:7-12. 31. Fauza DO, Fishman SJ, Mehegan K, Atala A. Videofetoscopically assisted fetal tissue engineering: skin replacement. J Pediatr Surg 1998;33:357-61. 32. Fauza DO, Marler JJ, Koka R, Forse RA, Mayer JE, Vacanti JP. Fetal tissue engineering:

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