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 prolife- ration 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 deve- lopment, 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 fe- atures of pluripotency when cultured in appro- priate 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