Fetal Liver-Derived Mesenchymal Stem Cell Engraftment After Allogeneic In Utero Transplantation into Rabbits

Abstract

Prenatal transplantation of genetically engineered mesenchymal stem cells (MSCs) might benefit prevention or treatment of early-onset genetic disorders due to the cells' intrinsic regenerative potential plus the acquired advantage from therapeutic transgene expression. However, a thorough assessment of the safety, accessibility, and behavior of these MSCs in the fetal environment using appropriate animal models is required before we can advance toward a clinical application. We have recently shown that fetal rabbit liver MSCs (fl-MSCs) have superior growth rate, clonogenic capability, and in vitro adherence and differentiation abilities compared with adult rabbit bone marrow MSCs. In this follow-up study, we report safe and widespread distribution of recombinant pSF-EGFP retrovirus-transduced fl-MSCs (EGFP + -fl-MSCs) in neonatal rabbit tissues at 10 days after fetal allogeneic transplantation through both intrahepatic and intra-amniotic administration. Conversely, a more restricted biodistribution pattern according to the route of administration was apparent in the young rabbits intervened at 16 weeks after fetal EGFP + -fl-MSC transplantation. Furthermore, the presence of these cells in the recipients' tissues, tracked with the reporter provirus, was inversely related to the developmental stage of the fetuses at the time of intervention. Long-term engraftment was confirmed both by fluorescence in situ hybridization analysis on touch tissue imprints using a chromosome Y-specific BAC probe, and by immunohistochemical localization of EGFP expression. Finally, there was no evidence of immune responses against the transplanted EGFP + -fl-MSCs or the EGFP transgenic product in the treated young rabbits. Thus, cell transplantation approaches using genetically engineered fetal MSCs may prove particularly valuable to frontier medical treatments for congenital birth defects in perinatology.

Engraftment Potential of Adipose Tissue-Derived Human Mesenchymal Stem Cells After Transplantation in the Fetal Rabbit

“…Analogously to that recently reported for in utero EGFP + -fl-MSC transplantation in the fetal rabbits [14], 2 casualties were documented among the EGFP + -ASC-treated pups due to nurturer rejection. The corresponding necropsies did not reveal any pathological sign in the deceased animals.…”

“…We intra-amniotically administered a unique dose of 2 · 10 5 EGFP + -ASCs (100 ml/fetus) into fetal rabbits at day E17 (control fetuses were injected with an identical volume of PBS) [14]. At E31 (14 days after intervention), pregnant rabbits were sacrificed (samples of gonads and uterus from the does and placenta from the pups were frozen for DNA extraction), and experimental pups were retrieved by visual identification after caesarean section, reanimated, and weighted.…”

“…Therefore, we screened transgenic EGFP expression of tissue sections positive for the presence of pWPT-EGFP provirus sequences from EGFP + -ASC-treated rabbits, as previously described [14]. However, we were not able to substantiate engraftment of xenogeneic EGFP + -ASCs into any postnatal in uterotreated rabbit tissue by immunohistochemistry.…”

“…For this purpose, we first analyzed whether young rabbits that underwent prenatal transplantation of EGFP + -ASCs overexpressing the reporter EGFP protein were able to develop a humoral immune response against the ASC-derived EGFP transgene product. A specific ELISA assay detecting the presence of antibodies against EGFP [14] was performed on sera harvested from young rabbits after in utero EGFP + -ASC delivery ( Fig. 2A).…”

“…We have recently evaluated the overall safety, biodistribution, engraftment capacity, and transgene expression of enhanced green fluorescent protein (EGFP)-expressing rabbit fetal liver MSCs (EGFP + -fl-MSCs) after allogeneic fetal transplantation into the pregnant rabbit model [14]. We highlighted the safety of fl-MSCs for fetal transplantation in the pregnant rabbit model, the efficiency, and persistence of fl-MSC engraftment and transgene expression in fetal organs, and the absence of immune reactions against both allogeneic fl-MSCs and transgenic EGFP after fetal transplantation.…”