Comparative Effects on Fetal Hematopoiesis and Placental Inflammation From Mesenchymal and Hematopoietic Stem Cells as Agents of Transamniotic Stem Cell Therapy (TRASCET) in a Syngeneic Model of Intrauterine Growth Restriction.

PURPOSE: We compared transamniotic stem cell therapy (TRASCET) using either mesenchymal (MSCs) or hematopoietic (HSCs) stem cells on fetal hematopoiesis in a syngeneic model of intrauterine growth restriction (IUGR). METHODS: Lewis dams exposed to cycling hypoxia (10.5% O2) in late gestation had their fetuses (n = 83) either receiving no intervention (untreated; n = 9), or intra-amniotic injections of either HSCs (HSC; n = 34), MSCs primed to an enhanced anti-inflammatory phenotype (primed-MSC; n = 28), or saline (sham; n = 12). Normal controls (n = 18) were also studied. Complete peripheral blood counts and placental ELISA for inflammation and angiogenesis markers were performed at term. RESULTS: Overall survival from hypoxia was 41% (34/83). Red blood count (RBC), hematocrit (Hct) and hemoglobin levels (Hb) were all significantly decreased from normal in all hypoxia groups. TRASCET with primed-MSC had significantly higher RBC, Hct, and Hb levels than sham (p = 0.01-0.03, pairwise), though not than untreated (which had no surgical blood loss). The HSC group had only significantly higher Hb levels than sham (p = 0.005). TRASCET with primed-MSC had significantly lower levels of placental TNF-α than sham (p = 0.04), but not untreated. CONCLUSIONS: MCSs seem more effective than HSCs in enhancing hematopoiesis when used as donor cells for TRASCET in a syngeneic model of IUGR. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

Bidirectional Feto-Maternal Traffic of Donor Mesenchymal Stem Cells Following Transamniotic Stem Cell Therapy (TRASCET).

PURPOSE: Transamniotic stem cell therapy (TRASCET) with mesenchymal stem cells (MSCs) has emerged experimentally as a potential treatment for different congenital diseases and maternal diseases of pregnancy. The broad applicability of TRASCET is predicated on hematogenous routing of donor MSCs via the placenta. We investigated whether donor MSC kinetics includes bidirectional traffic between the fetus and mother. METHODS: Eight time-dated dams had their fetuses (n = 96) divided in 4 groups on gestational day 17 (E17, term = E21). Groups populating one uterine horn received intra-amniotic injections (50 µL) of either donor amniotic fluid-derived MSCs (2×106 cells/mL) labelled with a firefly luciferase reporter gene (MSC-injected, n = 32), or of acellular luciferase (luciferase-injected, n = 26). Contra-lateral (CL) horn fetuses received no injection (MSC-CL, n = 20 and luciferase-CL, n = 18). At term, samples from 11 fetal anatomical sites from CL fetuses, along with placentas from all fetuses and maternal blood were screened for luciferase activity via microplate luminometry. RESULTS: Overall survival was 95 % (91/96). When controlled by the acellular injection, positive luciferase activity was observed in the placentas of all MSC-injected fetuses, confirming viability of the donor cells at term. When controlled by the acellular injection group, MSC-CL fetuses showed positive luciferase activity in the bone marrow, peripheral blood, brain and skin (p = <0.001-0.048). No luciferase activity was detected in any maternal blood sample. CONCLUSION: Amniotic fluid-derived MSCs can traffic between the fetus and mother in both directions after simple intra-amniotic injection, in a healthy rat model. This phenomenon must be considered in TRASCET performed in twin/multiple pregnancies. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

Morphometric, Developmental, and Anti-Inflammatory Effects of Transamniotic Stem Cell Therapy (TRASCET) on the Fetal Heart and Lungs in a Model of Intrauterine Growth Restriction.

Transamniotic stem cell therapy (TRASCET) with mesenchymal stem cells (MSCs) can attenuate placental inflammation and minimize intrauterine growth restriction (IUGR). We sought to determine whether MSC-based TRASCET could mitigate fetal cardiopulmonary effects of IUGR. Pregnant Sprague-Dawley dams were exposed to alternating 12-h hypoxia (10.5% O2) cycles in the last fourth of gestation. Their fetuses (n = 155) were divided into 4 groups. One group remained untreated (n = 42), while three groups received volume-matched intra-amniotic injections of either saline (sham; n = 34), or of syngeneic amniotic fluid-derived MSCs, either in their native state (TRASCET; n = 36) or "primed" by exposure to interferon-gamma and interleukin-1beta before administration in vivo (TRASCET-primed; n = 43). Normal fetuses served as additional controls (n = 30). Multiple morphometric and biochemical analyses were performed at term for select markers of cardiopulmonary development and inflammation previously shown to be affected by IUGR. Among survivors (75%; 117/155), fetal heart-to-body weight ratio was increased in both the sham and untreated groups (P < 0.001 for both) but normalized in the TRASCET and TRASCET-primed groups (P = 0.275, 0.069, respectively). Cardiac b-type natriuretic peptide levels were increased in all hypoxia groups compared with normal (P < 0.001), but significantly decreased from sham and untreated in both TRASCET groups (P < 0.0001-0.005). Heart tumor necrosis factor-alpha levels were significantly elevated in sham and TRASCET groups (P = 0.009, 0.002), but normalized in the untreated and TRASCET-primed groups (P = 0.256, 0.456). Lung transforming growth factor-beta levels were significantly increased in both sham and untreated groups (P < 0.001, 0.003), but normalized in both TRASCET groups (P = 0.567, 0.303). Similarly, lung endothelin-1 levels were elevated in sham and untreated groups (P < 0.001 for both), but normalized in both TRASCET groups (P = 0.367, 0.928). We conclude that TRASCET with MSCs decreases markers of fetal cardiac strain, insufficiency, and inflammation, as well as of pulmonary fibrosis and hypertension in the rodent model of IUGR.

Fetal Secretory IgA Delivery via Transamniotic Fetal Immunotherapy (TRAFIT) in a Rodent Model.

PURPOSE: We sought to determine the feasibility and routing kinetics of transamniotic fetal delivery of secretory immunoglobulin-A (SIgA), in a rodent model. METHODS: Fetuses (n = 94) from seven time-dated pregnant dams received intra-amniotic injections on gestational day 17 (E17, term = E21-22) of either saline (n = 15) or a solution of 1 mg/mL of ≥95% homogeneous human SIgA (n = 79). Animals were euthanized daily at E18-E21 for quantification of the IgA component by ELISA at gestational membranes, placenta, and select fetal anatomical sites against saline controls procured at term. Statistical analysis was by Mann-Whitney U-test. RESULTS: None of the saline-injected animals had detectable human IgA. SIgA-injected fetuses showed human IgA in the stomach aspirate, intestinal wall, lungs, liver, and serum at all time points. IgA levels were significantly higher in the gastric aspirate and in the intestine than in all other sites (p < 0.001 for both), with intestinal levels remaining stable through E18-E21 (p = 0.09-0.62 pairwise). Serum and placental levels were consistently low throughout, reaching near zero levels by E21. CONCLUSIONS: The chronology of exogenous secretory-IgA kinetics after intra-amniotic injection is suggestive of fetal uptake by ingestion, leading to consistent levels in the gastrointestinal tract. Transamniotic fetal immunotherapy (TRAFIT) with secretory-IgA may become a novel strategy for enhancing early mucosal immunity. LEVEL OF EVIDENCE: N/A (animal and laboratory study). TYPE OF STUDY: Animal and laboratory study.

Hematogenous Routing of Exogenous Messenger RNA Delivered Into the Amniotic Fluid.

INTRODUCTION: Therapies based on exogenous messenger RNA (mRNA) administration have emerged as a powerful novel strategy for the actual or potential treatment of an assortment of diseases, including congenital surgical pathologies. We sought to determine whether the minimally invasive transamniotic route could be an alternative for prenatal mRNA delivery. METHODS: Pregnant Sprague-Dawley dams underwent laparotomy followed by volume-matched intra-amniotic injections in all their fetuses (n = 120) of either a suspension of a custom firefly luciferase mRNA encapsulated by a lipid- and synthetic cationic polymer-based composite, or of a suspension of the same encapsulation components without mRNA, on gestational day 17 (E17; term = E21-22). On E18, E19, E20, and E21, samples from 14 fetal anatomical sites and maternal serum were procured for the screening of mRNA incorporation by host cells by measurement of luciferase activity via microplate luminometry. Statistical analysis was by Mann-Whitney U-test, including Bonferroni-adjustment. RESULTS: Overall survival was 87.5% (105/120). Controlled by the encapsulating composite without mRNA, luciferase activity was detected in the animals that received encapsulated mRNA in the following fetal annexes: amniotic fluid, amnion, chorion, umbilical cord, and placenta (P = 0.033 to <0.001), as well as in the following fetal sites: liver, stomach, intestines, and lungs (P = 0.043-0.002). CONCLUSIONS: Packaged exogenous mRNA can be incorporated by the fetus at least at select anatomical sites after simple intra-amniotic administration in a rodent model. The pattern and chronology of mRNA incorporation are compatible with transplacental hematogenous routing, as well as with fetal swallowing/aspiration. Further study of transamniotic mRNA administration is warranted.

Fetal Alloimmune Hemolytic Anemia (AHA) as a Potential Target for Transamniotic Fetal Immunotherapy (TRAFIT).

PURPOSE: Fetal alloimmune hemolytic anemia (AHA) resulting from maternal antibodies against fetal erythrocytes may require fetal administration of immunoglobulin-G (IgG) via invasive methods. IgG can reach the fetal circulation after transamniotic fetal immunotherapy (TRAFIT). We sought to both develop a model of AHA and to test TRAFIT as a potential treatment. METHODS: Sprague-Dawley fetuses (n = 113) received intra-amniotic injections on gestational-day 18 (E18, term = E21) of either saline (control; n = 40), anti-rat-erythrocyte antibodies (AHA; n = 37), or anti-rat-erythrocyte antibodies plus IgG (AHA + IgG; n = 36). At term, blood was procured for red blood count (RBC), hematocrit, or ELISA for inflammatory markers. RESULTS: There was no difference in survival [95% (107/113)] across groups (p = 0.87). Both hematocrit and RBC were significantly lower in the AHA group than controls (p < 0.001). Although still significantly lower than controls (p < 0.001), both hematocrit and RBC significantly increased in AHA + IgG group compared to AHA alone (p < 0.001). Pro-inflammatory TNF-α and IL1-ß were significantly elevated from controls in the AHA group, but not in AHA + IgG (p < 0.001-0.159). CONCLUSIONS: Intra-amniotic injection of anti-rat-erythrocyte antibodies can reproduce manifestations of fetal AHA, constituting a practical model of this disease. Transamniotic fetal immunotherapy with IgG reduces anemia in this model and may emerge as a new minimally invasive means of treatment. TYPE OF STUDY: Animal and laboratory study. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

Transamniotic stem cell therapy (TRASCET): An emerging minimally invasive strategy for intrauterine stem cell delivery.

Transamniotic stem cell therapy (TRASCET) is an emerging strategy for prenatal stem cell therapy involving the least invasive method described to date of delivering select stem cells to virtually any anatomical site in the fetus, including the blood and bone marrow, as well as to fetal annexes, including the placenta. Such broad therapeutic potential derives, to a large extent, from unique routing patterns following stem cell delivery into the amniotic fluid, which have commonalities with naturally occurring fetal cell kinetics. First reported experimentally only less than a decade ago, TRASCET has yet to be attempted clinically, though a first clinical trial appears imminent. Despite significant experimental advances, much promise and perhaps excessive publicity, most cell-based therapies have yet to deliver meaningful large-scale impact to patient care. The few exceptions typically consist of therapies based on the amplification of the normal biological role played by the given cells in their natural environment. Therein lays much of the appeal of TRASCET, in that it, too, is in essence a magnification of naturally occurring processes in the distinctive environment of the maternal-fetal unit. As much as fetal stem cells possess unique characteristics compared with other stem cells, so does the fetus when compared with any other age group, converging into a scenario that enables therapeutic paradigms exclusive to prenatal life. This review summarizes the diversity of applications and biological responses associated with the TRASCET principle.

Transamniotic Fetal Administration of Genetically Modified Hematopoietic Stem Cells Carrying a Human Transgene in a Syngeneic Rat Model.

Hematopoietic stem cell (HSC)-based gene therapy has already reached clinical reality in a few applications. Fetal administration of genetically modified HSCs has only been feasible to date through invasive and morbid methods. It has been recently shown that native donor HSCs can reach the fetal circulation and bone marrow after simple delivery into the amniotic fluid, at least in a syngeneic healthy model. We sought to determine whether the transamniotic route could also be a practical alternative for the fetal administration of genetically modified HSCs in a comparable model. Pregnant Lewis rat dams underwent volume-matched intra-amniotic injections in all their fetuses (n = 47) on gestational day 17 (E17; term = E21-22) of donor HSCs genetically modified using a custom lentiviral vector designed to constitutively express both a firefly luciferase reporter gene and a human adenosine deaminase (ADA) transgene. Donor HSCs consisted of syngeneic cells isolated from the amniotic fluid and phenotyped by flow cytometry. Fetuses were euthanized at term, when seven select sites relevant to HSC-based therapies were screened for either luciferase activity by luminometry or for the presence of human ADA mRNA by digital droplet polymerase chain reaction (ddPCR). Among survivors (30/47; 64%), positive luminescence and positive human ADA expression were detected in the bone marrow (respectively, 33% and 76%), liver (respectively, 11% and 81%), spleen (respectively, 11% and 67%), thymus (respectively, 33% and 67%), lungs (respectively, 44% and 86%), and brain (respectively, 22% and 90%). Nucleated peripheral blood cells were analyzed only by ddPCR, showing positive human ADA expression at 54%. We conclude that genetically modified HSCs can reach the fetal circulation and fetal bone marrow after simple intra-amniotic administration in a syngeneic rat model. Gene therapy by transamniotic HSC delivery may become a practicable, minimally invasive strategy for the prenatal treatment of select hemoglobinopathies, immunodeficiencies, and inherited metabolic disorders.

Early functional analysis on the pulmonary hemodynamic effects of Transamniotic Stem Cell Therapy (TRASCET) in the nitrofen model of congenital diaphragmatic hernia.

PURPOSE: Transamniotic stem cell therapy (TRASCET) with mesenchymal stem cells (MSCs) has been shown to impact pulmonary vascular development and remodeling in experimental congenital diaphragmatic hernia (CDH), with secondary structural cardiac effects. We sought to determine whether TRASCET has any functional impact on term fetal pulmonary hemodynamics in the nitrofen model. METHODS: Time-dated pregnant rat dams (n = 13) received nitrofen on gestational day 9 (E9) to induce fetal CDH. Fetuses (n = 155) were divided into three groups: untreated (n = 45), and two groups receiving volume-matched intra-amniotic injections on E17 of either saline (sham; n = 46), or a suspension of amniotic fluid-derived MSCs (afMSCs) (TRASCET; n = 64). Donor afMSCs were syngeneic, phenotyped by flow cytometry, and "primed" by exposure to interferon-gamma and interleukin-1beta prior to administration in vivo. At term (E21), fetuses underwent Doppler flow assessment at the mid-pulmonary artery and 4-chamber echocardiogram. Pulmonary vascular resistance was estimated by pulmonary artery acceleration time (PAAT), max velocity (MaxV) and velocity time integral (VTI). Cardiac function was assessed by global longitudinal strain (GLS) and ejection fraction (EF) using speckle analyses. Healthy fetuses (n = 11) served as additional controls. Statistical analysis was by the Mann-Whitney U test RESULTS: High resolution ultrasound data could be obtained from 8 to 13 fetuses per group. The PAAT and the PAAT normalized to cardiac cycle time were significantly improved by TRASCET compared to both untreated and sham-treated CDH (p = 0.004 to <0.001 in all pairwise comparisons). The flow profile sharpness (MaxV:VTI) was increased in untreated (p = 0.06) and sham (p = 0.01) groups but normalized by TRASCET (p<0.01). There was no difference in GLS between TRASCET and either the untreated or sham groups (p = 0.25 to p = 0.93). CONCLUSION: Transamniotic stem cell therapy improves pulmonary vascular resistance in early term fetuses in the Nitrofen model of congenital diaphragmatic hernia. Further focus on the functional pulmonary hemodynamic impact of this therapy is justified. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

Brain protection by transamniotic stem cell therapy (TRASCET) in a model of intrauterine growth restriction (IUGR).

PURPOSE: Transamniotic stem cell therapy (TRASCET) with mesenchymal stem cells (MSCs) has been shown experimentally to reverse some of the effects of intrauterine growth restriction (IUGR), apparently by attenuating placental inflammation. Neurodevelopmental deficits driven by neuroinflammation are major complications of IUGR. We sought to determine whether MSC-based TRASCET also mitigates inflammation in the fetal brain. METHODS: Pregnant Sprague-Dawley dams (n = 8) were exposed to alternating 12-hour hypoxia (10.5% O2) cycles from gestational day 15 (E15) until term (E21). One group remained untreated (n = 28 fetuses). Three groups received volume-matched intra-amniotic injections into all fetuses (n = 72) of either saline (sham; n = 19), or a suspension of amniotic fluid-derived MSCs, either in native state (TRASCET; n = 20), or primed by exposure to interferon-gamma (IFN-γ) and interleukin-1beta (IL-1ß) for 24 h prior to administration in vivo (TRASCET-Primed; n = 29). Donor MSCs were syngeneic Lewis rat cells phenotyped by flow cytometry. Normal fetuses served as controls (n = 20). Multiple analyses were performed at term, including ELISA in fetal brains for the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and IL-1ß. Statistical comparisons were by Wilcox-rank sum test, including Bonferroni-adjusted significance. RESULTS: Overall survival was 75% (88/116). Gross brain weights were significantly decreased from normal in both the untreated and sham groups (both p<0.001) and significantly increased in both TRASCET groups when compared to untreated and sham (p = 0.003 to <0.001). TRASCET-Primed led to significantly lower levels of TNF-α and IL-1ß compared to untreated (both p<0.001) and sham (p = 0.017 and p = 0.011, respectively). Non-primed TRASCET led to significantly lower levels of TNF-α and IL-1ß compared to untreated (p = 0.009 to <0.001), but not sham (p = 0.133 and p = 0.973, respectively). CONCLUSIONS: Transamniotic stem cell therapy with primed mesenchymal stem cells reverses some of the central nervous system effects of intrauterine growth restriction in a rat model, possibly by modulating neuroinflammation. TYPE OF STUDY: Animal and laboratory study. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

Transamniotic stem cell therapy (TRASCET) for intrauterine growth restriction (IUGR): A comparison between placental and amniotic fluid donor mesenchymal stem cells.

PURPOSE: Transamniotic stem cell therapy (TRASCET) with donor mesenchymal stem cells (MSCs) has been shown experimentally to reverse central effects of intrauterine growth restriction (IUGR). We sought to compare amniotic-fluid and placenta-derived MSCs (afMSCs and pMSCs, respectively) as TRASCET donor cells in a murine IUGR model. METHODS: Pregnant Sprague-Dawley dams (n=8) were exposed to alternating 12-hour hypoxia (10.5% O2) cycles, starting on gestational day 15 (E15; term=E21-22). On E17, fetuses (n=100) were divided into four groups. An untreated group had no further manipulations (n=24). Three groups received volume-matched intra-amniotic injections of either saline (sham; n=27), or suspensions of afMSCs (n=24), or pMSCs (n=25). Normal fetuses served as controls (n=21). All infused MSCs consisted of syngeneic Lewis rat cells phenotyped by flow cytometry and GFP-labeled. At term, fetal and placental morphometrics were calculated, and placental TNF-α levels were determined by ELISA. Statistical comparisons were by Fischer's T-test or Wilcoxon rank sum test (p≤0.05). RESULTS: Overall survival of the hypoxic groups was 83% (83/100). Compared to normal, maternal-adjusted fetal weights were significantly decreased in all hypoxia groups (pairwise p<0.001), however only the afMSC group showed higher adjusted-fetal weights than sham (p<0.001). Placental efficiency was decreased in untreated, sham, and pMSC groups (p<0.001-0.056) but normalized in the afMSC group (p=0.205). Maternal-adjusted placental weights were lower than normal in all hypoxia groups (p<0.001-0.045), except for the pMSC group (p=0.387). CONCLUSIONS: Amniotic fluid-derived mesenchymal stem cells are superior to their placenta-derived counterparts in transamniotic stem cell therapy for intrauterine growth restriction in a rat model. LEVEL OF EVIDENCE: Basic/Translational science.

Routing pathway of syngeneic donor hematopoietic stem cells after simple intra-amniotic delivery.

BACKGROUND: We sought to determine the pathway through which syngeneic hematopoietic stem cells (HSCs) delivered into the amniotic fluid can reach the fetal circulation. METHODS: Lewis rat fetuses were divided in two groups based on the content of intra-amniotic injections performed on gestational day 17 (E17; term=E21-22): either a suspension of luciferase-labeled syngeneic HSCs (n = 137), or acellular luciferase (n = 44). Samples from placenta, chorion, amnion, amniotic fluid, umbilical cord, and 8 fetal sites were procured at 5 daily time points thereafter until term for analysis. RESULTS: When controlled by acellular luciferase, donor HSCs were identified in the amnion, chorion, placenta, and amniotic fluid of fetuses receiving cells at all time points (p = 0.033 to <0.001), peaking first at the amnion and subsequently at the chorion and placenta. Cells could be detected in the fetal liver as early as day 1, progressively expanding to all the other fetal sites over time, in parallel to their increased presence in the chorion and placenta. CONCLUSIONS: The chronology of syngeneic donor hematopoietic stem cell trafficking after intra-amniotic injection is suggestive of controlled routing through the gestational membranes and placenta. Hematogenous donor cell routing is a constituent of transamniotic hematopoietic stem cell therapy, significantly expanding its potential applications.

Routing kinetics of human immunoglobulin-G after transamniotic fetal immunotherapy (TRAFIT) in a rodent model.

PURPOSE: The transamniotic route was recently discovered as a minimally invasive means of fetal immunoglobulin administration, however by unclear mechanisms. We sought to examine IgG routing after intra-amniotic delivery. METHODS: Sprague-Dawley fetuses (n = 78) received intra-amniotic injections of 15 mg/mL of human IgG on gestational-day 18 (E18; term=21 and 22 days). Amniotic fluid, amnion, chorion, placenta, fetal serum, liver, and stomach-aspirate samples were procured on E19, E20, and E21 for IgG quantification by ELISA. Statistical analysis was by median regression with Bonferroni-adjusted significance at p < 0.017. RESULTS: Human IgG was detected at all sampled sites across all time points, though at significantly higher levels in the gestational membranes and fetal serum than in the stomach aspirate and liver (p < 0.001 for both). Gestational membranes showed a daily decrease after injection, stabilizing by E20 and E21 (p = 0.792 to < 0.001). Placental levels were significantly lower at E21 than E19 (p = 0.010). Fetal serum showed the highest human IgG levels at term. CONCLUSIONS: The chronology of exogenous IgG kinetics after intra-amniotic injection is suggestive of direct placental transport leading to consistently high fetal serum levels, possibly combined with some fetal ingestion. Transamniotic fetal immunotherapy (TRAFIT) may become a practicable strategy for the prenatal treatment of select alloimmune disorders and infections. LEVEL OF EVIDENCE: N/A (Animal and Laboratory study). TYPE OF STUDY: Animal and Laboratory Study.

Intrauterine Growth Restriction (IUGR) as a potential target for transamniotic stem cell therapy.

BACKGROUND: We sought to determine whether intrauterine growth restriction (IUGR) could be a target for mesenchymal stem cell (MSC)-based transamniotic stem cell therapy (TRASCET). METHODS: Pregnant dams subjected to hypoxia (10.5% O2) cycles had their fetuses divided into four groups: untreated (n = 24) and three groups receiving volume-matched intra-amniotic injections of either saline (sham; n = 16), or suspensions of luciferase-labeled, syngeneic amniotic fluid-derived MSCs that were either native (TRASCET-unprimed; n = 29), or primed by exposure to IFNγ and IL-1ß (TRASCET-primed; n = 31). Normal fetuses served as additional controls (n = 22). Multiple analyses were performed at term. RESULTS: Compared to normal, fetal weights were significantly decreased in all hypoxia groups (p = 0.002 to <0.001), except for TRASCET-primed. Placental efficiency (fetal/placental weight) was significantly decreased in all hypoxia groups (p = 0.002 to <0.001), but normalized in both TRASCET groups. A significant increase in metrial expression of IFNγ in both the untreated and sham groups (p = 0.04 to 0.02) was reversed only in the TRASCET-primed group. Luciferase DNA was present in both TRASCET groups' placentas. CONCLUSIONS: Transamniotic stem cell therapy with primed mesenchymal stem cells reverses some of the effects of intrauterine growth restriction in a rat model. Further study into this novel approach for the treatment of this disease is warranted. LEVEL OF EVIDENCE: N/A (Animal and Laboratory Study).

Passive perinatal immunotherapy via transamniotic antibody delivery.

PURPOSE: We sought to determine whether the amniotic cavity/fluid could be an attainable route of administration of therapeutic antibodies to the fetus/neonate. METHODS: Time-dated pregnant dams (n = 9) received volume-matched intra-amniotic injections of either saline (n = 29), or different concentrations of a human IgG that lacked homology with rodents: 5 mg/mL (n = 28); 10 mg/mL (n = 28); or 15 mg/mL (n = 24). At term, the presence of the IgG was quantified by ELISA in the serum, bone marrow, spleen, thymus, and brain of all neonates, and in the maternal serum. Statistical analysis was by median regression with significance set at Bonferroni-adjusted p<0.008. RESULTS: Overall fetal survival was 83% (90/109), with no difference between the groups. Human IgG was detected in the serum, bone marrow, spleen, thymus, and brain of all fetuses for all three injected concentrations, but not in the saline injected controls (p<0.001). A dose dependent relationship between injection concentration and final IgG load was noted in the bone marrow, spleen, and thymus (p = 0.004 to <0.001). Human IgG was also detected in maternal serum. CONCLUSIONS: IgG antibodies can reach high levels in the fetal/neonatal circulation after simple intra-amniotic administration in a healthy rodent model. Transamniotic fetal immunotherapy (TRAFIT) may become a practicable strategy for the perinatal management of select diseases. LEVEL OF EVIDENCE: N/A (animal and laboratory study) TYPE OF STUDY: Animal and laboratory study.

Transamniotic Stem Cell Therapy for Experimental Congenital Diaphragmatic Hernia: Structural, Transcriptional, and Cell Kinetics Analyses in the Nitrofen Model.

PURPOSE: We examined select pulmonary effects and donor cell kinetics after transamniotic stem cell therapy (TRASCET) in a model of congenital diaphragmatic hernia (CDH). METHODS: Pregnant dams (n = 58) received nitrofen on gestational day 9.5 (E9) to induce fetal CDH. Fetuses (n = 681) were divided into 4 groups: untreated (n = 99) and 3 groups receiving volume-matched intra-amniotic injections on E17 of either saline (n = 142), luciferase-labeled amniotic fluid-derived mesenchymal stem cells (afMSCs; n = 299), or acellular recombinant luciferase (n = 141). Pulmonary morphometry, quantitative gene expression of pulmonary vascular tone mediators, or screening for labeled afMSCs were performed at term (E22). Statistical comparisons were by Mann-Whitney U-test, nested ANOVA, and Wald test. RESULTS: TRASCET led to significant downregulation of endothelial nitric oxide synthase and endothelin receptor-A expressions compared to both untreated and saline groups (both p < 0.001). TRASCET also led to a significant decrease in arteriole wall thickness compared to the untreated group (p < 0.001) but not the saline group (p = 0.180). Donor afMSCs were identified in the bone marrow and umbilical cord (p = 0.035 and 0.015, respectively, vs. plain luciferase controls). CONCLUSIONS: The effects of TRASCET in experimental CDH appear to be centered on the pulmonary vasculature and to derive from circulating donor cells.

Enhancement of transamniotic stem cell therapy for spina bifida by genetic engineering of donor mesenchymal stem cells with an Fgf2 transgene.

BACKGROUND/PURPOSE: We examined whether engineered overexpression of fibroblast growth factor-2 (Fgf2) in donor mesenchymal stem cells (MSCs) could enhance spina bifida coverage induced by transamniotic stem cell therapy (TRASCET). METHODS: Pregnant Sprague-Dawley dams (n = 24) exposed to retinoic acid for induction of fetal spina bifida were divided in three groups. An untreated group had no further manipulations. Two groups received volume-matched intra-amniotic injections into all fetuses (n = 157) of either amniotic fluid-derived MSCs (afMSC; n = 85) or afMSCs transduced with an Fgf2 transgene (Fgf2-afMSC; n = 72) on gestational day 17 (term=21-22 days). Defect coverage was categorized at term by histology and pan-cytokeratin immunohistochemistry. Statistical coverage comparisons were by logistic regression. RESULTS: Among 84 survivors with isolated spina bifida, 71 had definitive histology. Defect coverage rates in both the afMSC (38.5%) and Fgf2-afMSC (73.3%) groups were statistically significantly higher than in the untreated group (10%; p<0.001 for both). There was a significantly higher coverage rate in the Fgf2-afMSC group compared with the afMSC group (p = 0.025). CONCLUSIONS: Fgf2 overexpression in donor mesenchymal stem cells increases defect coverage rates in a rodent model of transamniotic stem cell therapy for spina bifida. Genetic engineering of donor cells is a promising strategy for the enhancement of this emerging therapy.