Amniotic Fluid Stem Cells: an Ideal Resource for Therapeutic Application in Bone Tissue Engineering

Home , Amniotic stem cells

European Review for Medical and Pharmacological Sciences 2016; 20: 2884-2890 Amniotic fluid stem cells: an ideal resource for therapeutic application in bone tissue engineering

A. PANTALONE1, I. ANTONUCCI2, M. GUELFI1, P. PANTALONE3, F.G. USUELLI4, L. STUPPIA2, V. SALINI1

1Orthopaedic and Traumatology Division, Department of Medicine and Science of Aging, University G. d’Annunzio, Chieti-Pescara, Chieti, Italy 2Laboratory of Molecular Genetics, Department of Psychological, Health and Territorial Sciences, School of Medicine and Health Sciences, “G. d’Annunzio University” Chieti-Pescara; Ce.S.I. -MeT, “G. d’Annunzio” University, Chieti-Pescara, Italy 3Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, UK 4Foot and Ankle Unit, IRCCS Galeazzi, Milan, Italy

Abstract. – OBJECTIVE: Skeletal diseases, also in other clinical conditions, like osteonecro- both degenerative and secondary to trauma, in- sis of the femoral head, osteogenesis imperfecta fections or tumors, represent an ideal target for and hypophosphatasia. The current cure for bone regenerative medicine and in the last years, stem cells have been considered as good candidates repair is autologous bone graft, but this approach for in vitro and in vivo bone regeneration. To date, is limited by non-structural integration of autolo- several stem cell sources, such as adult mesen- gous fragments, and cell-based therapies may be chymal stem cells, embryonic stem cells (ESCs) particularly effective for the treatment in patien- and induced pluripotent stem cells (iPSCs), have ts with reduced presence of endogenous stem or shown significant osteogenic potential. progenitor cells because of advanced age. MATERIALS AND METHODS: In this narrative review, we analyze the possible advantages of As a consequence, the treatment of trauma- the use of AFSCs in the treatment of skeletal dis- tic and degenerative bone defects represents eases, especially through the application of tis- one of the main targets of regenerative medi- sue engineering and biomaterials. cine and tissue engineering, which are novel RESULTS: Among the different sources of stem multidisciplinary approaches aimed to recon- cells, great attention has been recently devoted to struct tissues or organs in order to replace amniotic fluid-derived stem cells (AFSC) character- ized by high renewal capacity and ability to differ- damaged and injured parts of the body. Since 5 entiate along several different lineages. the pioneristic studies of Haynesworth et al , CONCLUSIONS: Due to these features, AFSCs where the stem cells (Figure 1), in particular represent an interesting model for regenerative Bone Marrow-Mesenchymal Stem Cells (BM- medicine, also considering their low immunoge- MSCs), were differentiated in bone, cartilage nicity and the absence of tumor formation after transplantation in nude mice. and other musculoskeletal tissues, MSCs have been largely used with three-dimensional scaf- Key Words: folds in order to repair or replace missing or Bone regeneration, Amniotic fluid, Amniotic flu- damaged tissues to a state as close as possi- id-derived mesenchymal stem cells, Biotechnology. ble to their native architecture and function6. However, the use of adult BM shows some li- Introduction mitations, such as the low frequency of MSCs (about 0.001-0.01% of nucleated cells) and the Skeletal diseases, like critical bone defects due invasivity of the BM harvesting from patien- to trauma, infections or tumors, as well as atrophic ts. Therefore, also different cell types, such as nonunions and osteoporosis represent a major embryonic, fetal or adult stem cells, and gene- challenge for orthopaedic reconstructive surge- tically engineered cell lines, have been tested ons, requiring a large amount of bone regenera- for their ability to replace damaged cells and tion for their effective treatment and resolution1-4. to restore the tissue function after transplanta- A compromised regenerative process is observed tion7. Embryonic Stem Cells (ESCs) are able to

2884 Corresponding Author: Matteo Guelfi, MD; e-mail: [email protected] Amniotic fluid stem cells

Figure 1. Sources and differentiation of MSCs. differentiate in cells belonging to all the three In recent years, different reports have de- embryonic layers, being thus able to produce monstrated the presence in human amniotic flu- virtually each different cell type. However, id (AF) of stem cells (AFSCs) able to differen- ESCs induce the formation of teratomas when tiate into multiple lineages and to produce cell injected in nude mice, and their use raises types inclusive of all embryonic germ layers8-11, several ethical concerns. On the other hand, suggesting the possible usefulness of these cel- adult stem cells have been largely used in the ls for therapeutic purposes12-15. clinical setting, despite their limited proliferation and differentiation ability. Autologous Amniotic Fluid-Derived Stem Cells cells would represent the ideal transplanta- Human AF samples can be easily obtained du- tion source, being not rejected by the immune ring the process of amniocentesis from women system and allowing to avoid the use of im- undergoing prenatal diagnosis (16th-19th week of munosuppressant drugs. However, these cel- pregnancy) and contains a heterogeneous popu- ls show limited “ex vivo” expansion abilities, lation of cell types originating from embryonic particularly when collected from patients with and extra-embryonic tissues, classified as epithe- end-stage organ disease7. The use of allogenic lioid (E-type) cells, amniotic fluid specific (AF- cells would be preferred in these cases, but this type) cells, and fibroblastic (F-type) cells8-16. For may require the creation of cell banks contai- this reason, cell populations in AF show great ning a large number of samples from different diversity and variation among amniocentesis donors immunologically matched with the po- samples from different donors, time of gestation, tential patients. and cultivation17.

2885 A. Pantalone, I. Antonucci, M. Guelfi, P. Pantalone, F.G. Usuelli, L. Stuppia, V. Salini

vation of the X-chromosome in female samples was also demonstrated. Taken together, these data suggest a germ cell origin of AFSCs. It has also been hypothesized that during early development, PGC-derived from epiblast could be the founder of these cells. Similar results were obtained by Moschidou’s group22, that confirmed the presence of cells of germ origin in human first-trimester AF and the same group suggested that also mid-trimester AFSCs c-KIT+, show features of pluripotency when cultured in appropriate media23. In last years, AFSCs have been used by several groups in preclinical studies to treat different pathological conditions such as Figure 2. AFSCs of second passage. ischemic brain injury with encouraging resul- ts24-29. In particular, the outcome of Tajiri et al studies30,31 supported the idea that immunomo- About 1% of cells from AF (approximately 2.7 dulatory and paracrine effects are predominant x 105 cells from each sample) are considered stem in the therapeutic properties of these cells. Final- cells18 and in the majority of studies so far repor- ly, the discovery of several MSC-like cell in AF ted, AFSCs ( 2) have been isolated using a se- has allowed to isolate and select amniotic fluid lection based on the expression of the surface an- mesenchymal stem cells (AFMSC) by means of tigen c-Kit (CD117) present on ESCs, primordial the culture methodology32. AFMSC present mul- germ cells and many somatic stem cells (c-Kit+ tipotency, self-renewal, low immunogenicity, AFSCs)9. However, other authors18 demonstrated anti-inflammatory, non-tumorigenicity proper- that AFSCs can be obtained also by cultivating ties, have a normal karyotype and minimal ethi- AF cells without any kind of previous selection cal problem. Taken together, these cells may be (unselected AFSCs). appropriate sources of mesenchymal stem cells The interest raised by AFSCs is related to for regenerative medicine, as an alternative to the presence of specific features making these embryonic stem cells (ESCs)18,33-36. cells different both from pluripotent ESCs and multipotent adult stem cells. About 90% of AF- AFS as available Source of Osteogenic SCs express specific markers of ESCs, such as Progenitor Cells Oct-4 and TERT19,20. However, unlike ESCs, AF- The ability of AFSCs to differentiate in osteo- SCs are not tumorigenic after transplantation in genic precursors has been demonstrated by several mice. Thus, AFSCs represent a new class of stem authors, and their functional activity was confir- cells with properties of plasticity intermediate med by the capacity to produce in vivo minerali- between embryonic and adult stem cell types. In zed matrix and bone tissue11,36-42. The efficiency of order to verify the presence of features of plu- this process has been demonstrated by clonogenic ripotency in human second trimester AFSCs, mineralization assays which have evidenced that Antonucci et al21 have investigated the ability 85% of AFSCs versus 50% of MSCs are capable of these cells to form in vitro three-dimensional of forming osteogenic colonies. Osteogenic diffe- aggregates, known as embryoid bodies (EBs), rentiation of AFSCs can be improved through the and to express specific genes of embryonic stem use of specific culture protocols. Antonucci et al39 cells (ESCs) and primordial germ cells (PGCs). have demonstrated that osteogenic differentiation EBs showed positivity for alkaline phosphatase of AFSCs can be obtained in a very short time (AP) staining and specific markers of pluripo- through a single step protocol and after 30 days tency (OCT4, NANOG and SOX2) and the three from the withdrawal of AF samples, osteoblasts germ layers. The most important result concerns progenitor display a complete expression of oste- the presence of specific markers of ESCs (such ogenic markers (COL1, ONC, OPN, OCN, OPG, as FGF4 and DAPPA4), as well as markers typi- BSP, Runx2). Interestingly, differentiated AFSCs cal of PGCs and, in particular, genes involved cells are able to growth on scaffolds and surfaces in early stages of germ cell development (Fra- commonly used in orthopedic surgery, as eviden- gilis, Stella, Vasa, c-Kit, Rnf17). Finally, inacti- ced by several reports; in particular, these cells

2886 Amniotic fluid stem cells even after osteogenic differentiation maintain a expressed in proliferating and osteo-differentia- good ability to proliferate on titanium screws40 ted cells and huAFMSCs could be considered a commonly used in orthopaedic implantology. potential osteogenic model to study in-vitro cell These attractive results suggest their possible ap- responses to calcitonin (and other members of the plication in cell therapy of bone injuries40. Sun et calcitonin family). al43 demonstrated that osteoblastic differentiation of AFSCs and their induction to generate mine- AFS Based Therapies for Bone ralized tissue can be obtained also by the use of Tegeneration culture protocols based on the use of bone mor- Due to their excellent ability to differentiate phogenic protein 7 (BMP-7). Compared to hM- into osteogenic precursors, AFSCs can be con- SCs, AFSCs showed a stronger responsiveness to sidered as a very promising tool in the area of BMP-7, in particular when cultured on synthetic bone regeneration. De Coppi et al11 early demon- NF scaffolds providing a more favorable microe- strated that, after subcutaneous implantation nvironment and enhancing their osteoblastic dif- into immunodeficient mice of osteogenically differentiation in vitro and bone formation in vivo43. ferentiated AFSCs embedded in an alginate/col- In this view, the usefulness of the use of specific lagen scaffold, it was possible to observe ectopic scaffolds during AFSCs culture to improve their bone formation, suggesting that these cells could ability to differentiate into osteogenic precursors be used to engineer bone grafts for the repair of has been clearly demonstrated also by Peister et bone defects. A practical approach to the use al44 which demonstrated that the “in vitro” pre-dif- of AFSCs for postnatal sternal repair has been ferentiation of AFSCs in porous medical-grade reported by Steigman et al48, who isolated rab- poly-e-caprolactone (mPCL) scaffolds produced bit AFSCs, cultured on biodegradable nanofib- seven times more mineralized matrix after subcu- ers and then implanted them into full-thickness taneous in vivo implantation in a rat model. The- sternal defects. Two months after implantation, se results demonstrate the potential of these cells in vivo imaging modalities confirmed chest clo- to produce 3D mineralized bioengineered con- sure and bone formation. This study concluded structs in vitro and in vivo suggesting their use- that engineered bone tissues can be a viable al- fulness for functional repair of large bone defects. ternative for sternal repair and that AFSCs can Recently, it has been studied calcium-sensing re- be a practical cell source for engineered chest ceptor (CaSR) expression in hAFMSCs and the wall reconstruction. activity of calcimimetic R-568 during in vitro The potential of AFSCs to synthesize mineral- osteogenesis. The results demonstrated that CaSR ized extracellular matrix within different porous is expressed in these cells and positively corre- scaffolds of collagen, poly-D, L-lactic acid (PDL- lates with osteogenic markers. This information LA), and silk fibroin was investigated by Maraldi is important because clarifies the mechanisms of et al49, who induced osteoblastic differentiation hAFMSC osteogenesis and could provide additio- of these cells by using both two-dimensional nal molecular basis for the use of calcimimetics in cultures and three-dimensional scaffolds (colla- bone regenerative medicine45. In addition, it has gen, fibroin, and PDLLA). The effect of AFSCs been observed that the canonical Wnt/ß-catenin pre-differentiation in scaffolds on the subsequent signaling pathway appears to trigger huAFMSC bone formation in vivo was determined in a rat osteoblastogenesis, since during early phases of subcutaneous model, evidencing a higher osteo- osteogenic differentiation, the expression of Di- genic differentiation and mineralized extracellu- shevelled-2 (Dvl-2), of the non-phosphorylated lar matrix production. Authors suggested that fi- form of ß-catenin, and the phosphorylation of broin may represent an effective scaffold material glycogen synthase kinase-3ß (GSK3ß) at serine 9 for functional repair of critical size bone defects. were upregulated. The modulation of Wnt signa- Rodrigues et al50 investigated whether the differ- ling may represent a novel approach to direct cells entiation stage of AFSC could improve bone re- towards a more definite differentiation. However, generation in a rat model of critical sized femoral future studies are awaited to assess the validity of defect. AFSCs were seeded onto a starch-poly this strategy46. Finally, Morabito’s group47 studied (ε-caprolactone) (SPCL) scaffold and cultured in the effects of calcitonin on huAFMSCs during order to obtain undifferentiated cells, cells com- osteogenic differentiation, in terms of the physio- mitted to the osteogenic phenotype (2 weeks of logical role of calcitonin in bone homeostasis. The culture) and “osteoblast-like” cells (3 weeks of data obtained showed that calcitonin receptor was culture). Constructs composed of AFSC-SPCL

2887 A. Pantalone, I. Antonucci, M. Guelfi, P. Pantalone, F.G. Usuelli, L. Stuppia, V. Salini scaffolds from each differentiation stage were even after in vitro transplantation. However, then implanted into critical sized femoral defects, also other specific features of AFSCs suggest and the formation of new bone was examined that these cells could represent in a next future by micro-CT imaging and histological analysis the gold standard for regenerative medicine in of constructs retrieved at 4 and 16 weeks after the orthopaedic field. In fact, a further element implantation. The best results in terms of com- supporting the usefulness of AFSCs for allo- plete repair of the defect were showed by animals genic transplantation is provided by their low treated with SPCL scaffolds seeded with osteo- immunogenicity, since only a small fraction genically committed AFSC, in which it was pos- of these cells are slightly positive for antigens sible to evidence the presence of blood vessels in HLA-DR (MHC class II). AFSCs are resistant the inner sections of the scaffolds, suggesting the to rejection due to the expression of immuno- potential of differentiated AFSCs inducing bone suppressive factors such as CD59 (protectin), regeneration and angiogenesis in non-union bone which inhibits the complement membrane at- defects50. A further evidence of the usefulness of tack complex and prevent complement from AFSCs in regenerative medicine has been provid- damaging cells, and HLA-G, which plays a key ed by Turner et al51 in a study carried out on New role in immune tolerance in pregnancy52. More- Zealand rabbits in which a full-thickness diplo- over, AFSCs have been demonstrated as able to ic nasal bone defect had been induced. Animals inhibit the proliferation of T lymphocytes53. As were treated with size-matched implants of elec- a consequence, AFSCs offer an advantage over trospun biodegradable nanofibers with or without cells derived from other sources, being able allogeneic AFSs cultivated in osteogenic medium. to survive after allogeneic transplant without Post-mortem analysis evidenced similar levels of using immunosuppressive therapy. Moreover, defect radiodensity in the two groups, but extra- due to these specific features, low cost proto- cellular calcium levels were significantly higher cols to isolate AFSCs could be established, in in engineered grafts than in acellular implants. order to create banks containing all MHC im- Authors concluded that AF derived engineered munotypes, which could be used for allogenic bone could represent a useful tool in perinatal clinical applications in the orthopaedic field. craniofacial reconstruction.

Conflicts of interest Conclusions The authors declare that they have no conflicts of interest concerning this article. No financial support has The use of adult stem cells in regenerative been received by the authors for the preparation of this medicine may alleviate ethical and availabili- manuscript. ty concerns, with the additional advantages, in some cases, to allow autologous grafts. The References demonstrated presence of stem cells within AF have raised great interest due to the large ac- cessibility of these cells by means of routine 1) Dimitriou R, Jones E, McGonagle D, Giannoudis PV. amniocentesis, their ability to differentiate in Bone regeneration: current concepts and future several cell lineages, the absence of tumorigeni- directions. BMC Medicine 2011; 9: 66-76. Kon E, Filardo G, Roffi A, Di Martino A, Hamdan M, city after transplantation and the lack of ethical 2) L. Pasqual L, Merli ML, Marcacci M. Bone regene- problems related to their use. Compared to em- ration with mesenchymal stem cells. Clin Cases bryonic stem cells, amniotic stem cells can be Miner Bone Metab 2012; 9: 24-27. obtained without destroying human embryos, 3) Quarto R, Mastrogiacomo M, Cancedda R, Kutepov thus solving much of the ethical controversy. SM, Mukhachev V, Lavroukov A, Kon E, Marcacci M. Stem cells from AF could be useful both for Repair of large bone defects with the use of auto- personalized cells supply for newly born chil- logous bone marrow stromal cells. N Engl J Med dren and for banking cells to be used for thera- 2001; 344: 385-386. peutic cell transplantation in immunologically 4) Waese EY, Kandel RA, Stanford WL. Application of stem cells in bone repair. Skeletal Radiol 2008; matched recipients. As reported above, AFSCS 37: 601-608. have demonstrated to be able to differentiate 5) Haynesworth SE, Goshima J, Goldberg VM, Caplan into osteogenic precursors under different cul- AI. Characterization of cells with osteogenic po- ture conditions, and to produce mineral matrix tential from human marrow. Bone 1992; 13: 81-88.

2888 Amniotic fluid stem cells

6) Khan WS, Longo UG, Adesida A, Denaro V. Stem niotic fluid as rich source of mesenchymal stromal cell and tissue engineering applications in or- cells for transplantation therapy. Cell Transplant thopaedics and musculoskeletal medicine. Stem 2010; 20: 789-795. Cells Int 2012; 2012: 403170. 21) Antonucci I, Pietro RD, Alfonsi M, Centurione 7) Hipp J, Atala A. Sources of stem cells for regenera- MA, Centurione L, Sancilio S, Pelagatti F, D Amico tive medicine. Stem Cell Rev 2008; 4: 3-11. MA, Baldassarre AD, Piattelli A, Tetè S, Palka G, Borlongan CV, Stuppia L. 8) Prusa AR, Hengstschlager M. Amniotic fluid cells Human second-trime- and human stem cell research: a new connection. ster amniotic fluid cells are able to create em- Med Sci Monit 2002; 8: 253-257. bryoid body-like structures in vitro and to show typical expression profiles of embryonic and Prusa AR, Marton E, Rosner M, Bernaschek G, Hen- 9) primordial germ cells. Cell Transplant 2014; 23: gstschlager M. Oct-4-expressing cells in human 1501-1515. amniotic fluid: a new source for stem cell resear- Moschidou D, Drews K, Eddaoudi A, Adjaye J, De ch? Hum Reprod 2003; 18: 1489-1493. 22) Coppi P, Guillot PV. Molecular signature of hu- Fauza D. 10) Amniotic fluid and placental stem cells. man amniotic fluid stem cells during fetal de- Best Pract Res Clin Obstet Gynaecol 2004; 18: velopment. Curr Stem Cells Res Ther 2013; 8: 877-891. 73-81. De Coppi P, Bartsch G Jr, Siddiqui MM, Xu T, Santos 11) 23) Moschidou D, Mukherjee S, Blundell MP, Drews K, erin ostoslavsky erre nyder CC, P L, M G, S AC, S EY, Jones GN, Abdulrazzak H, Nowakowska B, Phool- oo urth oker tala Y JJ, F ME, S S, A A. Isolation of am- chund A, Lay K, Ramasamy TS, Cananzi M, Nettersheim niotic stem cell lines with potential for therapy. Nat D, Sullivan M, Frost J, Moore G, Vermeesch JR, Fisk Biotechnol 2007; 25: 100-106. NM, Thrasher AJ, Atala A, Adjaye J, Schorle H, De 12) In ‘t Anker PS, Scherjon SA, Kleijburg-van der Keur C, Coppi P, Guillot PV. Valproic acid confers functio- Noort WA, Claas FH, Willemze R, Fibbe WE, Kanhai nal pluripotency to human amniotic fluid stem cel- HH. Amniotic fluid as a novel source of mesen- ls in a transgene-free approach. Mol Ther 2012; chymal stem cells for therapeutic transplantation. 20: 1953-1967. Blood 2003; 102: 1548-1549. 24) Sartore S, Lenzi M, Angelini A, Chiavegato A, Gaspa- 13) Holden C. Stem cells. Versatile stem cells without rotto L, De Coppi P, Bianco R, Gerosa G. Amniotic the ethical baggage? Science 2007; 315: 170. mesenchymal cells autotransplanted in a porcine 14) Kunisaki SM, Freedman D, Fauza DO. Fetal tracheal model of cardiac ischemia do not differentiate to reconstruction with cartilaginous grafts engine- cardiogenic phenotypes. Eur J Cardiothorac Surg ered from mesenchymal amniocytes. J Pediatr 2005; 28: 677-684. Surg 2006; 41: 675-682. 25) Flores AI, Gómez-Gómez GJ, Masedo-González Á, Martínez-Montiel MP 15) Da Sacco S, De Filippo RE, Perin L. Amniotic fluid as . Stem cell therapy in a source of pluripotent and multipotent stem cells inflammatory bowel disease: A promising the- for organ regeneration. Curr Opin Organ Tran- rapeutic strategy? World J Stem Cells 2015; 7: splant 2011; 16: 101-105. 343-351. 16) Zhang S, Geng H, Xie H, Wu Q, Ma X, Zhou J, Chen 26) Ekblad Å, Qian H, Westgren M, Le Blanc K, Fossum F. The heterogeneity of cell subtypes from a pri- M, Götherström C. Amniotic fluid--a source for mary culture of human amniotic fluid. Cell Mol Biol clinical therapeutics in the newborn? Stem Cells Lett 2010; 15: 424-439. Dev 2015; 24: 1405-1414. 17) Pipino C, Pierdomenico L, Di Tomo P, Di Giuseppe F, 27) Ramachandra DL, Shaw SS, Shangaris P, Loukogeor- Cianci E, D’Alimonte I, Morabito C, Centurione L, gakis S, Guillot PV, Coppi PD, David AL. In utero the- Antonucci I, Mariggiò MA, Di Pietro R, Ciccarelli R, rapy for congenital disorders using amniotic fluid Marchisio M, Romano M, Angelucci S, Pandolfi A. stem cell. Front Pharmacol 2015; 6: 39. Molecular and phenotypic characterization of hu- 28) Li Y, Gu C, Xu W, Yan J, Xia Y, Ma Y, Chen C, He X, man amniotic fluid-derived cells: a morphological Tao H. Therapeutic effects of amniotic fluid-de- and proteomic approach. Stem Cells Dev 2015; rived mesenchymal stromal cells on lung injury 24: 1415-1428. in rats with emphysema. Respir Res 2014; 15: 18) Antonucci I, Pantalone A, Tete S, Salini V, Borlongan 120. CV, Hess D, Stuppia L. Amniotic fluid stem cells: a 29) Danieli P, Malpasso G, Ciuffreda MC, Cervio E, Cal- promising therapeutic resource for cell-based re- villo L, Copes F, Pisano F, Mura M, Kleijn L, de Boer generative therapy. Current Pharmaceutical Desi- RA, Viarengo G, Rosti V, Spinillo A, Roccio M, Gnec- gn 2012; 18: 1846-1863. chi M. Conditioned medium from human amniotic 19) Mauro A, Turriani M, Ioannoni A, Russo V, Martelli mesenchymal stromal cells limits infarct size and A, Di Giacinto O, Nardinocchi D, Berardinelli P. Iso- enhances angiogenesis. Stem Cells Transl Med lation, characterization, and in vitro differentiation 2015; 4: 448-458. of ovine amniotic stem cells. Vet Res Commun 30) Tajiri N, Acosta S, Glover LE, Bickford PC, Jacotte Si- 2010; 34: 25-28. mancas A, Yasuhara T, Date I, Solomita MA, Antonuc- 20) Antonucci I, Stuppia L, Kaneko Y, Yu S, Tajiri N, Bae ci I, Stuppia L, Kaneko Y, Borlongan CV. Intravenous EC, Chheda SH, Weinbren NL, Borlongan CV. Am- grafts of amniotic fluid- derived stem cells induce

2889 A. Pantalone, I. Antonucci, M. Guelfi, P. Pantalone, F.G. Usuelli, L. Stuppia, V. Salini

endogenous cell proliferation and attenuate beha- rived stem cells. Alcohol Clin Exp Res 2010; 34: vioral deficits in ischemic stroke rats. PLoS One 1714-1722. 2012; 7: e43779. 43) Sun H, Feng K, Hu J, Soker S, Atala A, Ma PX. 31) Tajiri N, Acosta S, Portillo-Gonzales GS, Aguirre D, Osteogenic differentiation of human amniotic flu- Reyes S, Lozano D, Pabon M, Dela Peña I, Ji X, Ya- id-derived stem cells induced by bone morpho- suhara T, Date I, Solomita MA, Antonucci I, Stuppia genetic protein-7 and enhanced by nanofibrous L, Kaneko Y, Borlongan CV. Therapeutic outcomes scaffolds. Biomaterials 2010; 31: 1133-1139. of transplantation of amniotic fluid-derived stem 44) Peister A, Deutsch ER, Kolambkar Y, Hutmacher DW, cells in experimental ischemic stroke. Front Cell Guldberg RE. Amniotic fluid stem cells produce ro- Neurosci 2014; 8: 227. bust mineral deposits on biodegradable scaffolds. 32) Loukogeorgakis SP, De Coppi P. Stem cells from am- Tissue Engineering Part A 2009: 15: 3129-3138. niotic fluid–Potential for regenerative medicine. 45) Pipino C, Di Tomo P, Mandatori D, Cianci E, Lanuti Best Pract Res Clin Obstet Gynaecol 2015 Sep P, Cutrona MB, Penolazzi L, Pierdomenico L, Lamber- 10. tini E, Antonucci I, Sirolli V, Bonomini M, Romano 33) Joerger-Messerli MS, Marx C, Oppliger B, Mueller M, M, Piva R, Marchisio M, Pandolfi A. Calcium sen- Surbek DV, Schoeberlein A. Mesenchymal stem sing receptor activation by calcimimetic R-568 in cells from wharton’s jelly and amniotic fluid. human amniotic fluid mesenchymal stem cells: Best Pract Res Clin Obstet Gynaecol 2015 correlation with osteogenic differentiation. Stem Sep. Cells Dev 2014; 23: 2959-2971. 34) Savickiene J, Treigyte G, Baronaite S, Valiuliene G, 46) D’Alimonte I, Lannutti A, Pipino C, Di Tomo P, Pier- Kaupinis A, Valius M, Arlauskiene A, Navakauskiene domenico L, Cianci E, Antonucci I, Marchisio M, Ro- R. Human amniotic fluid mesenchymal stem cells mano M, Stuppia L, Caciagli F, Pandolfi A, Ciccarelli from second- and third-trimester amniocentesis: R. Wnt signaling behaves as a “master regulator” differentiation potential, molecular signature, and in the osteogenic and adipogenic commitment of proteome analysis. Stem Cells Int 2015; 2015: human amniotic fluid mesenchymal stem cells. 319238. Stem Cell Rev 2013; 9: 642-654. 35) Wang KH, Kao AP, Chang CC, Lin TC, Kuo TC. Upre- 47) Morabito C, D’Alimonte I, Pierdomenico L, Pipino C, gulation of Nanog and Sox-2 genes following Guarnieri S, Caprara GA, Antonucci I, Ciccarelli R, ectopic expression of Oct-4 in amniotic fluid me- Marchisio M, Pandolfi A, Mariggiò MA. Calcito- senchymal stem cells. Biotechnol Appl Biochem nin-induced effects on amniotic fluid-derived me- 2015; 62: 591-597. senchymal stem cells. Cell Physiol Biochem 2015; 36) Kim L, Lee Y, Kim H, Hwang KJ, Kwon HC, Kim SK, 36: 259-273. Cho DJ, Kang SG, You J. Human amniotic fluid-de- 48) Steigman SA, Ahmed A, Shanti RM, Tuan TS, Valim rived stem cells have characteristics of multipo- C, Fauza DO. Sternal repair with bone grafts engi- tent stem cells. Cell Prolif 2007; 40: 75-90. neered from amniotic mesenchymal stem cells. J 37) Pappa KI, Anagnou NP. Novel sources of fetal stem Pediatr Surg 2009; 44: 1120-1126. cells: where do they fit on the developmental con- 49) Maraldi T, Riccio M, Resca E, Pisciotta A, La Sala GB, tinuum? Regen Med 2009; 4: 423-433. Ferrari A, Bruzzesi G, Motta A, Migliaresi C, Marzona 38) Tsai MS, Lee JL, Chang YJ, Hwang SM. Isolation of L, De Pol A. Human amniotic fluid stem cells see- human multipotent mesenchymal stem cells from ded in fibroin scaffold produce in vivo mineralized second-trimester amniotic fluid using a novel matrix. Tissue Engineering Part A 2011; 17: 2833- two-stage culture protocol. Hum Reprod 2004; 2843. 19:1450-1456. 50) Rodrigues MT, Lee BK, Lee SJ, Gomes ME, Reis RL, 39) Antonucci I, Iezzi I, Morizio E, Mastrangelo F, Pan- Atala A, Yoo JJ. The effect of differentiation stage talone A, Mattioli-Belmonte M, Gigante A, Salini V, of amniotic fluid stem cells on bone regeneration. Calabrese G, Tetè S, Palka G, Stuppia L. Isolation of Biomaterials 2012; 33: 6069-6078. osteogenic progenitors from human amniotic fluid 51) Turner CG, Klein JD, Gray FL, Ahmed A, Zurakowski using a single step culture protocol. BMC Biotech- D, Fauza DO. Craniofacial repair with fetal bone nol 2009; 16: 9. grafts engineered from amniotic mesenchymal 40) Antonucci I, Pantalone A, De Amicis D, D’Onofrio S, stem cells. J Surg Res 2012; 178: 785-790. Stuppia L, Palka G, Salini V. Human amniotic fluid 52) Walther G, Gekas J, Bertrand OF. Amniotic stem stem cells culture onto titanium screws: a new cells for cellular cardiomyoplasty: promises and perspective for bone engineering. J Biol Regul premises. Catheter Cardiovasc Interv 2009; 73: Homeost Agents 2009; 23: 277-279. 917-924. Chen Q, Xiao P, Chen JN, Cai JY, Cai XF, Ding H, Pan 41) 53) Sessarego N, Parodi A, Podestà M, Benvenuto F, Mo- YL. AFM studies of cellular mechanics during osteo- gni M, Raviolo V, Lituania M, Kunkl A, Ferlazzo G, genic differentiation of human amniotic fluid-derived Bricarelli FD, Uccelli A, Frassoni F. Multipotent me- stem cells. Anal Sci 2010; 26: 1033-1037. senchymal stromal cells from amniotic fluid: solid 42) Hipp JA, Hipp JD, Atala A, Soker S. Ethanol alters perspectives for clinical application. Haematologi- the osteogenic differentiation of amniotic fluid-de- ca 2008; 93: 339-346.

2890