Amniotic stem cells reprogramming for tissue regeneration in animal model prenatal myelomeningocele repair.

AIM OF THE STUDY Neural tube defects (NTDs), including spina bifida (with/without myelomeningocele), are caused by the failure of closure of the neural tube during gestation. Current preventive strategies are mainly based on pharmacologic supplementation during pregnancy, while current therapy is essentially postnatal surgical closure with patches. However, fetal surgery emerges as potential treatment option for NTDs. In addition, autologous stem cell-based approach could come in help with fetal surgery in the repair of the NTDs. The aim of this study is to prove the feasibility of the process of isolation and characterization of stem cells collected from amniotic fluid (AFSCs) of ovine animal model of NTDs in order to improve the knowledge regarding the NTDs repair in utero. METHODS Through a median longitudinal laparotomy and hysterotomy, fetal lambs at 12 week of gestation underwent open creation of a MMC defect through a posterior laminectomy encompassing lumbar vertebrae 1–4. We collected amniotic fluid during this surgery and 4 weeks later. AFSCs were isolated from amniotic fluids and cultured in growth medium composed of DMEM 15% FCS and 5 ng/mL FGF [De Coppi et al., 2007]. After approximately 7 days confluent cells were detached by 1% Trypsin and frozen for subsequent analysis. MAIN RESULTS Our results indicate that it is possible to isolate a large number of cells (ca. 1 million cells/sample) from fetal sheep amniotic fluid. We have collected samples at week 12, 16, 20. For each time point, an average of 70% of samples produced a stable clone of AFSCs. Preliminary results indicate that they have potency and self-renewal and thus, they are stem cells. CONCLUSION AND FUTURE PERSPECTIVE Our findings highlight the possibility to isolate AFSCs from ovine animal model of NTD and set up the basis for future cell therapy-based therapeutics. Characterization of AFSC subpopulations by FACS, qPCR and WB to better identify the best candidate for NTD treatment is still in progress. In addition, AFSC reprogramming using Yamanaka’s factors is currently under investigation. These preclinical studies are needed to increase the feasibility of new regenerative therapeutic approaches for NTDs.