Trans-Amniotic Stem Cell Therapy (TRASCET) Minimizes Chiari-II Malformation in Experimental Spina Bifida
Purpose: Prenatal surgical repair of spina bifida minimizes the incidence and severity of the associated Chiari-II malformation, however it can only be offered to a relatively small cohort of fetuses and not without possible complications. Amniotic fluid-derived mesenchymal stem cells (afMSCs) have been shown to elicit variable degrees of coverage of experimental spina bifida after simple intra-amniotic injection in large numbers. We sought to determine whether this therapeutic strategy could also have any impact on the Chiari-II malformation in this setting.
Methods: Following IACUC approval, 11 time-dated pregnant Sprague-Dawley dams exposed to retinoic acid for the induction of fetal neural tube defects were divided in two groups: one (n=6) had no further manipulations and another (n=5) received volume-matched intra-amniotic injections of a suspension of 2x106 cells/mL of afMSCs blindly in all fetuses on gestational day 17 (term=21-22 days). Infused afMSCs consisted of syngeneic Lewis rat cells with mesenchymal identity confirmed by flow cytometry, labeled with green fluorescent protein (GFP). Animals were killed before term, when fetuses were divided into three groups: untreated controls with isolated spina bifida (n=21); isolated spina bifida treated with trans-amniotic delivery of afMSCs (n=28); and normal controls (n=13). Analyses included magnetic resonance imaging (MRI) with a high resolution (sub-millimeter) scanner and histology. The Chiari-II malformation was assessed on MRI by computer-generated specific angular and linear measurements of brainstem and cerebellar placement in relation to the basooccipital bone and the base of the skull, respectively (figures). Statistical analyses were by Pearson χ2, Fisher's exact test, and ANOVA with Bonferroni comparisons (2-tailed P<0.05).
Results: As expected, there was a statistically significant increase in the proportion of fetuses with variable degrees of coverage of the spina bifida by a rudimentary skin confirmed histologically in the afMSC-treated group (P<0.001). Overall, there were statistically significant differences across the groups in linear and angular measurements of brainstem placement (P<0.001), with the untreated group displaying the highest caudal displacement. All pairwise comparisons of these parameters were statistically significant, with P=0.014 between treated and normal controls in angular brainstem (caudal) displacement and P<0.001 for all other angular and linear pairwise comparisons. Differences in cerebellar placement were also noted, albeit less pronounced, with P<0.001 overall and significance in most pairwise comparisons, except between treated and untreated groups (P=0.10). Donor afMSCs were identified in 71% (20/28) of fetuses in the treated group via immunohistochemistry for GFP.
Conclusions: Induced coverage of spina bifida by concentrated trans-amniotic delivery of amniotic mesenchymal stem cells does not completely reverse, however does minimize Chiari-II malformation in the retinoic acid rodent model. Trans-amniotic administration of amniotic-derived mesenchymal stem cells may become a practical alternative or adjuvant in the prenatal management of spina bifida.