Transamniotic Stem Cell Therapy Modulates Uterine Natural Killer Cell Activity in the Hypoxia Model of Intrauterine Growth Restriction.

Intrauterine growth restriction (IUGR) pathophysiology is driven by abnormal uterine natural killer cell (uNK) activity leading to placental dysfunction. Transamniotic stem cell therapy (TRASCET) with mesenchymal stem cells (MSCs) can improve experimental IUGR by mechanisms not fully understood. We sought to examine TRASCET's effects in downstream products of uNKs in a model of IUGR: 15 Sprague-Dawley dams were exposed to alternating hypoxia (10.5% O2) from gestational day 15 (E15) until term (E21). Their fetuses (n = 189) were divided into four groups. One group remained untreated (n = 52), whereas three groups received volume-matched intraamniotic injections of either saline (sham, n = 44) or a suspension of amniotic fluid-derived MSCs, either in their native state (TRASCET, n = 50) or "primed" to an enhanced antiinflammatory phenotype (TRASCET-Primed, n = 43). Normal fetuses served as controls (n = 33). At term, various analyses were performed, including ELISA for surrogates of placental inflammation and uNK activity. Statistical comparisons included Bonferroni-adjusted criterion. Overall survival from hypoxia was 74% (140/189). Placental efficiency was lower in untreated and sham but normalized in both TRASCET groups (P < 0.01-0.47). Interleukin-17, a stimulator of uNKs, was elevated from normal in all groups (P < 0.01 for all). Interferon-gamma, released from activated uNKs, was elevated in all groups except sham but lower than the untreated in both TRASCET groups (P ≤ 0.01-0.06). Tumor necrosis factor-alpha, also produced by uNKs, was elevated in untreated and sham (P < 0.01 for both), but normalized by TRASCET (P = 0.05) and even lowered from normal in TRASCET-Primed (P < 0.01). Vascular endothelial growth factor, also released by uNKs, was elevated in untreated and sham but lower than normal in both TRASCET groups (P < 0.01 for all). We conclude that TRASCET with MSCs modulates the activity of placental uNKs in experimental IUGR, with distinct effects on their downstream products. This mechanistic insight may inform the development of novel strategies for the management of this disease.

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.

Barriers in translating stem cell therapies for neonatal diseases.

Over the last 20 years, stem cells of varying origin and their associated secretome have been investigated as a therapeutic option for a myriad of neonatal models of disease, with very promising results. Despite the devastating nature of some of these disorders, translation of the preclinical evidence to the bedside has been slow. In this review, we explore the existing clinical evidence for stem cell therapies in neonates, highlight the barriers faced by researchers and suggest potential solutions to move the field forward.

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.

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.

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).

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.

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).

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.

Fetal hematogenous routing of a donor hematopoietic stem cell line in a healthy syngeneic model of transamniotic stem cell therapy.

BACKGROUND/PURPOSE: In utero administration of hematopoietic stem cells (HSCs) has a variety of actual or potential clinical applications but is hindered by invasive, morbid administration techniques. We sought to determine whether donor HSCs could reach the fetal circulation after simple intra-amniotic delivery in a syngeneic rat model of transamniotic stem cell therapy (TRASCET). METHODS: Pregnant Lewis rat dams underwent volume-matched intra-amniotic injections in all fetuses (n = 90) on gestational day 17 (E17; term=E21-22) of a suspension of commercially available syngeneic Lewis rat HSCs labeled with luciferase (n = 37 fetuses) or an acellular suspension of recombinant luciferase (n = 53). HSC phenotype was confirmed by flow cytometry. Fetuses were euthanized at term for screening of luciferase activity at select anatomical sites. Statistical comparisons were by Fisher's exact test. RESULTS: Among survivors (47/90; 52.2%), donor HSCs were identified selectively in the placenta (p = 0.003), umbilical cord (p < 0.001), bone marrow (p < 0.001), thymus (p = 0.009), bowel (p = 0.003), kidney (p = 0.022), and skin (p < 0.001) when compared with acellular luciferase controls. CONCLUSIONS: Donor hematopoietic stem cells undergo hematogenous routing and can reach the fetal bone marrow after simple intra-amniotic delivery in a syngeneic rat model. Transamniotic stem cell therapy may become a practicable, minimally invasive strategy for the prenatal administration of these cells.

Initial Mechanistic Screening of Transamniotic Stem Cell Therapy in the Rodent Model of Spina Bifida: Host Bone Marrow and Paracrine Activity.

PURPOSE: Transamniotic stem cell therapy (TRASCET) with mesenchymal stem cells (MSCs) can induce spina bifida coverage with neoskin. We initiated a mechanistic analysis of this host response. METHODS: Pregnant dams (n = 28) exposed to retinoic acid to induce fetal spina bifida were divided into an untreated group and 2 groups receiving intra-amniotic injections on gestational day 17 (E17; term = E21-22) of either amniotic fluid-derived MSCs (afMSCs; n = 105) or saline (n = 107). Gene expressions of multiple paracrine and cell clonality markers were quantified at term by RT-qPCR at the defect and fetal bone marrow. Defects were examined histologically for neoskin coverage. Comparisons were by Mann-Whitney U tests and logistic regression. RESULTS: Defect coverage was associated with significant downregulation of both epidermal growth factor (Egf; p = 0.031) and fibroblast growth factor-2 (Fgf-2; p = 0.042) expressions at the defect and with significant downregulation of transforming growth factor-beta-1 (Tgfb-1; p = 0.021) and CD45 (p = 0.028) expressions at the fetal bone marrow. CONCLUSIONS: Coverage of experimental spina bifida is associated with local and bone marrow negative feedback of select paracrine factors, as well as increased relative mesenchymal stem cell activity in the bone marrow. Further analyses informed by these findings may lead to strategies of nonsurgical induction of prenatal coverage of spina bifida.

Hematogenous Donor Cell Routing Pathway After Transamniotic Stem Cell Therapy.

Donor mesenchymal stem cells (MSCs) have been documented in fetal and maternal circulations after plain intra-amniotic injection, with diverse therapeutic effects. We sought to determine the pathway of this unique cell kinetic route. Rat fetuses (n = 226) were divided into two groups based on the content of intra-amniotic injections performed on gestational day 17 (E17): either a concentrated suspension of luciferase-labeled syngeneic amniotic fluid-derived MSCs (afMSCs; n = 111), or acellular luciferase (n = 115). Samples from placenta, chorion, amnion, amniotic fluid, stomach fluid, peripheral blood, and umbilical cord were procured at five daily time points thereafter until term (E18-22) for luminometry. In addition, 53 sets of fresh gestational membranes (chorion/amnion combined) from nonmanipulated term fetuses were secured to transwell inserts for in vitro analysis of MSC migration using luciferase-labeled afMSCs. Statistical analyses included the Mann-Whitney U-test, Wald test, nonlinear regression modeling, and Fisher's exact test. In vivo, luciferase activity was observed in the amnion, chorion, and placenta of fetuses receiving cells, but not in those receiving acellular luciferase (P < 0.001). There was a consistent nonlinear age-dependent relationship of luciferase activity between the amnion, chorion, and placenta following a parabolic bimodal pattern characterized by significantly higher early preterm (E18) and late-term (E22) activities (P < 0.001), with no differences between E21 and E22 (P = 0.12). In vitro, the presence of cells was documented by luminometry in 21/53 (39.6%) of the assays, in suspension and/or attached to the plastic substrate, and within all screened gestational membrane sets, irrespective of stimuli with collagen coating or fetal bovine serum. We conclude that, after intra-amniotic injection, donor MSCs undergo controlled cell routing, as opposed to passive clearance. Transgestational membrane transport appears to constitute the path for donor cells to reach the placenta, a known gateway to the fetal circulation, significantly expanding the potential applications of transamniotic stem cell therapy.

Postnatal fate of donor mesenchymal stem cells after transamniotic stem cell therapy in a healthy model.

PURPOSE: We sought to examine donor mesenchymal stem cell (MSC) fate after birth following transamniotic stem cell therapy (TRASCET) in a healthy model. METHODS: Lewis rat fetuses (n = 91) were divided into two groups based on the content of volume-matched intraamniotic injections performed on gestational day 17 (term = 21-22 days): either a suspension of amniotic fluid-derived MSCs (afMSCs) labeled with luciferase (n = 38) or acellular luciferase only (n = 53). Infused afMSCs consisted of syngeneic Lewis rat cells phenotyped by flow cytometry. Samples from 14 anatomical sites (heart, lung, brain, liver, spleen, pancreas, bowel, kidney, thyroid, skin, skeletal muscle, thymus, peripheral blood and bone marrow) from survivors were screened for luciferase activity 16 days after birth. Statistical analysis was by logistic regression and the Wald test (p < 0.05). RESULTS: Overall survival was 32% (29/91). When controlled by the acellular luciferase injections, donor afMSCs were not identified at any anatomical site in any neonate as measured by relative light units (all p > 0.05). Donor afMSC viability was confirmed in term placentas. CONCLUSIONS: Donor mesenchymal stem cells are not detectable in the neonate after intraamniotic injection in a normal syngeneic rodent model. This finding suggests that clinical trials of transamniotic stem cell therapy may be amenable to regulatory approval. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

Congenital diaphragmatic hernia as a potential target for transamniotic stem cell therapy.

PURPOSE: We sought to determine whether TRASCET could impact congenital diaphragmatic hernia (CDH). METHODS: Twelve pregnant dams received Nitrofen on gestational day 9.5 (E9; term = 22 days) to induce fetal CDH. Fetuses were divided into three groups: untreated (n = 31) and two groups receiving volume-matched intraamniotic injections of either saline (n = 37) or a suspension of 2 × 106 cells/mL of amniotic fluid-derived mesenchymal stem cells (afMSCs; n = 65) on E17. Animals were euthanized at term. Expression of fibroblast growth factor-10 (FGF-10), vascular endothelial growth factor-A (VEGF-A), and surfactant protein-C (SPC) was quantified by qRT-PCR. Statistical analysis was by the Mann-Whitney U test with Bonferroni adjusted criterion (p ≤ 0.01). RESULTS: Among survivors with CDH (n = 27/133), the TRASCET group showed significant downregulation of FGF-10 and VEGF-A gene expressions compared to the untreated (p < 0.001 for both) and saline groups (p = 0.005 and p = 0.004, respectively). SPC expression was higher in the TRASCET group compared to the untreated group (p = 0.01), but not the saline group (p = 0.043). Lung laterality had minimal impact on these comparisons. CONCLUSIONS: Transamniotic stem cell therapy affects select processes of lung development in experimental congenital diaphragmatic hernia. Further scrutiny into this novel therapy as a potential component of the prenatal management of this disease is warranted. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

Donor mesenchymal stem cell kinetics after transamniotic stem cell therapy (TRASCET) in a rodent model of gastroschisis.

PURPOSE: We sought to comprehensively scrutinize donor mesenchymal stem cell kinetics following transamniotic stem cell therapy (TRASCET) in experimental gastroschisis. METHODS: A gastroschisis was surgically created in 102 rat fetuses at gestation day 18 (term = 22 days), immediately followed by volume-matched amniotic injections of either amniotic fluid mesenchymal stem cells (afMSCs) labeled with a luciferase reporter gene (n = 58), or luciferase protein alone (n = 44). Samples from multiple anatomical sites from survivors were screened for luciferase activity via microplate luminometry at term. Statistical analysis included Mann-Whitney U-test, Wald test, and kappa coefficient (p < 0.05). RESULTS: Overall survival was 42% (43/102), with no significant difference between the two groups (p = 0.82). When controlled by acellular luciferase, donor afMSCs were identified selectively in the placenta (p < 0.001) and bowel (p = 0.005), independently of the dams (respectively, p < 0.001 and p = 0.041). Bowel homing was documented exclusively in areas exposed to the amniotic cavity. There was no mutual correlation between placental and bowel homing (kappa = -0.02; p = 0.91). CONCLUSIONS: Amniotic mesenchymal stem cells home to specific sites after TRASCET in the setting of gastroschisis. Placental homing and intestinal homing are central yet seemingly independent constituents of cell trafficking, suggesting that both direct amniotic seeding and hematogenous routing take place. LEVEL OF EVIDENCE: N/A (animal and laboratory study).