Hester F Shieh, MD, Sarah A Tracy, MD, Charles R Hong, MD, Alexander V Chalphin, MD, Azra Ahmed, BS, Lucas Rohrer, BS, David Zurakowski, PhD, Dario O Fauza, MD, PhD
Boston Children's Hospital, Boston, MA, USA

Purpose: Transamniotic stem cell therapy (TRASCET) with amniotic or placental mesenchymal stem cells (MSCs) has been shown to induce partial or complete skin coverage of spina bifida in a rodent model. Clinical translation of this emerging therapy hinges on its efficacy in larger animal models. We sought to study TRASCET in a model requiring intra-amniotic injections 60 times larger than those performed in the rat. Methods: New Zealand rabbit fetuses (n=65) with surgically created spina bifida at gestational day 22-23 (term 32-33 days) were divided into three groups. One group (untreated) had no further manipulations. Two groups received volume-matched intra-amniotic injections of either saline or a suspension of 2x106 cells/mL of amniotic fluid MSCs (afMSCs) at the time of operation. Infused afMSCs consisted of banked heterologous rabbit afMSCs with mesenchymal identity confirmed by flow cytometry, labeled with green fluorescent protein (GFP). Animals were killed before term. Defect coverage was categorized only if the local presence of a distinctive rudimentary neoskin was confirmed histologically. Statistical comparisons were by logistic regression and the likelihood ratio test (two-tailed P<0.05).  Results: Expectedly for this model, overall survival was 29%, with no statistically significant differences between groups (27-33%; P=0.690-0.891 in pairwise comparisons). Among survivors with spina bifida (n=19), there were statistically significant higher rates of defect coverage (all partial) in the afMSC group when compared with the saline and untreated groups (0-50%; P=0.022-0.036). There were no significant differences in coverage rates between the saline and untreated groups (P=1.00). Donor afMSCs were identified locally via immunohistochemistry for GFP, though sparsely and not in the neoskin. Conclusions: Concentrated intra-amniotic injection of amniotic mesenchymal stem cells can induce partial coverage of experimental spina bifida in a surgical leporine model. Transamniotic stem cell therapy may become a feasible and accessible strategy in the prenatal management of spina bifida.