Congenital Diaphragmatic Hernia as a Potential Target for Transamniotic Stem Cell Therapy: Comprehensive Donor Cell Homing and Pulmonary Morphometric Analyses

Purpose: Transamniotic stem cell therapy (TRASCET) can impact select processes of pulmonary development in experimental congenital diaphragmatic hernia (CDH), particularly in the lung vasculature. We sought to examine pulmonary morphometrics and donor cell kinetics after TRASCET in experimental CDH.

Methods: Following IACUC approval, forty-three pregnant dams received Nitrofen (2,4-dichlorophenyl p-nitrophenyl-ether) orally on gestational day 9.5 (E9; term=22 days) to induce fetal CDH. Fetuses were divided into four groups: untreated animals undergoing no further manipulations (n=59) and three groups receiving volume-matched intra-amniotic injections on E17 of either saline (n=103), a suspension of amniotic fluid-derived mesenchymal stem cells (afMSCs) labeled with luciferase (TRASCET; n=231), or an acellular suspension of recombinant luciferase (n=141). Normal fetuses (n=16) served as additional controls. Infused afMSCs consisted of syngeneic rat cells phenotyped by flow cytometry. Animals were euthanized at term for either pulmonary morphometry, or screening for labeled afMSC presence at twelve anatomical sites. Statistical comparisons were by Wald test (two-tailed p<0.05) and nested ANOVA (Bonferroni-adjusted p<0.008).

Results: Among 103 survivors with CDH, morphometric and homing analyses were performed in 56 and 47 fetuses, respectively. TRASCET led to a significant decrease in arteriole wall thickness compared to the untreated group (p<0.001; figure), but this did not reach significance against the saline group with the number of subjects available (p=0.180; figure). There were no significant differences in any of the five parameters of alveolarization compared between untreated and both treatment groups (figure). Donor afMSCs were identified selectively in the bone marrow and umbilical cord (p=0.035 and 0.015, respectively, vs. plain luciferase controls).

Conclusions: The effects of transamniotic stem cell therapy in experimental congenital diaphragmatic hernia appear to be centered on the pulmonary vasculature and to derive from circulating donor cells. Further scrutiny into the impact of this therapy on pulmonary arterial pressures and function is warranted.