Long-term engraftment and phenotype correction has been difficult to achieve in humans after in utero stem cell transplantation mainly because of allogeneic rejection. Autologous cells could be obtained during gestation from the amniotic fluid with minimal risk for the fetus and the mother. Using a sheep model, we explored the possibility of using amniotic fluid mesenchymal stem cells (AFMSCs) for autologous in utero stem cell/gene therapy. We collected amniotic fluid (AF) under ultrasound-guided amniocentesis in early gestation pregnant sheep (n = 9, 58 days of gestation, term = 145 days). AFMSCs were isolated and expanded in all sampled fetal sheep. Those cells were transduced using an HIV vector encoding enhanced green fluorescent protein (GFP) with 63.2% (range 38.3-96.2%) transduction efficiency rate. After expansion, transduced AFMSCs were injected into the peritoneal cavity of each donor fetal sheep at 76 days under ultrasound guidance. One ewe miscarried twin fetuses after amniocentesis. Intraperitoneal injection was successful in the remaining 7 fetal sheep giving a 78% survival for the full procedure. Tissues were sampled at postmortem examination 2 weeks later. PCR analysis detected GFP-positive cells in fetal tissues including liver, heart, placenta, membrane, umbilical cord, adrenal gland, and muscle. GFP protein was detected in these tissues by Western blotting and further confirmed by cytofluorimetric and immunofluorescence analyses. This is the first demonstration of autologous stem cell transplantation in the fetus using AFMSCs. Autologous cells derived from AF showed widespread organ migration and could offer an alternative way to ameliorate prenatal congenital disease.
Prenatal gene therapy aims to deliver genes to cells and tissues early in prenatal life, allowing correction of a genetic defect, before long-term tissue damage has occurred. In contrast to postnatal gene therapy, prenatal application can target genes to a large population of dividing stem cells, and the smaller fetal size allows a higher vector-to-target cell ratio to be achieved. Early-gestation delivery may allow the development of immune tolerance to the transgenic protein which would facilitate postnatal repeat vector administration if needed. Targeting particular organs will depend on manipulating the vector to achieve selective tropism and on choosing the most appropriate gestational age and injection method for fetal delivery. Intra-amniotic injection reaches the skin, and other organs that are bathed in the fluid however since gene transfer to the lung and gut is usually poor more direct injection methods will be needed. Delivery to the liver and blood can be achieved by systemic delivery via the umbilical vein or peritoneal cavity. Gene transfer to the central nervous system in the fetus is difficult but newer vectors are available that transduce neuronal tissue even after systemic delivery.
Reports of a rare form of cerebral venous sinus thrombosis with profound thrombocytopenia have emerged following introduction of the adenovirus-vectored coronavirus disease 2019 (COVID-19) vaccines. Between March and June 2021, seven cases of refractory vaccine-induced immune thrombotic thrombocytopenia were referred to our institution for mechanical thrombectomy. The condition of 1 patient deteriorated during interhospital transfer, and the remaining 6 underwent successful recanalization. No procedure-related adverse events were reported. At the time of this writing, 3 patients have been discharged with a good functional outcome (mRS 0-1), one required rehabilitation for mild dysarthria and vocal cord palsy (mRS 3), and 2 have died due to severe mass effect. Our anecdotal experience suggests that endovascular therapy may be safe and effective in reducing thrombus burden in selected cases of postvaccination cerebral venous sinus thrombosis.ABBREVIATIONS: COVID-19 ¼ coronavirus disease 2019; CVST ¼ cerebral venous sinus thrombosis; HIT ¼ heparin-induced thrombocytopenia; ICH ¼ intracerebral hemorrhage; PF ¼ platelet factor; VITT ¼ vaccine-induced immune thrombotic thrombocytopenia
Our data indicate that sildenafil citrate does not ameliorate L-NAME-induced IUGR, and in the doses utilized in this study might even have a synergistic negative effect on pup birth weight.
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