In vitro cell culture of amniotic fluid keratinocytes on amniotic membrane: the ideal tissue for repairing skin ulcers.

OBJECTIVE: The amniotic fluid contains a large population of stem keratinocytes demonstrating minimal immunological rejection. Recent evidence suggests that stem cells from the amniotic fluid can be employed in the field of tissue engineering. In this work we identified precursors of the epithelial cells and expanded them in vitro. MATERIALS AND METHODS: After collecting samples of amniotic fluid and separating the cells via centrifugation, we seeded a portion of these cells in selection media to analyze the proliferation of epithelial cells. The stem cells precursors of keratinocytes were identified through specific markers. The expression of these markers was evaluated by immunofluorescence and reverse transcription polymerase chain reaction (PCR). RESULTS: The stem cells demonstrated 90% confluence, after undergoing proliferation in the selection medium for 15 days. Most of these cells tested positive for the keratinocyte-specific markers, but negative for stem cell specific markers. Of note, the identity of the keratinocytes was well established even after several subcultures. CONCLUSIONS: These results suggested that it is feasible to isolate and expand differentiated cell populations in the amniotic fluid from precursor cells. Furthermore, amniotic membranes can be utilized as scaffolds to grow keratinocytes, which can be potentially exploited in areas of skin ulcer transplantation and tissue engineering interventions.

Survivability of rabbit amniotic fluid-derived mesenchymal stem cells post slow-freezing or vitrification.

This work aimed to evaluate the effect of two distinct cryopreservation procedures - conventional slow-freezing and vitrification, on survivability and mesenchymal marker expression stability of rabbit amniotic fluid-derived mesenchymal stem cells (rAF-MSCs). Cells at passage 2 were slowly frozen, using 10% of dimethylsulfoxide, or vitrified, using 40% of ethylene glycol, 0.5 M sucrose and 18% Ficoll 70. After three months storage in liquid nitrogen, viability, chromosomal stability, ultrastructure, surface and intracellular marker expression and differentiation potential of cells were evaluated immediately post-thawing/warming and after additional culture for 48-72 h. Our results showed decreased (P ≤ 0.05) viability of cells post-thawing/warming. However, after additional culture, the viability was similar to those in fresh counterparts in both cryopreserved groups. Increase (P ≤ 0.05) in the population doubling time of vitrified cells was observed, while doubling time of slow-frozen cells remained similar to non-cryopreserved cells. No changes in karyotype (chromosomal numbers) were observed in frozen/vitrified AF-MSCs, and histological staining confirmed similar differentiation potential of fresh and frozen/vitrified cells. Analysis of mesenchymal marker expression by qPCR showed that both cryopreservation approaches significantly affected expression of CD73 and CD90 surface markers. These changes were not detected using flow cytometry. In summary, the conventional slow-freezing and vitrification are reliable and effective approaches for the cryopreservation of rabbit AF-MSCs. Nevertheless, our study confirmed affected expression of some mesenchymal markers following cryopreservation.

Amniotic fluid volume at presentation with early preterm prelabor rupture of membranes and association with severe neonatal respiratory morbidity.

OBJECTIVE: Amniotic fluid volume (AFV) plays an important role in early fetal lung development, and oligohydramnios in early pregnancy is associated with pulmonary hypoplasia. The aim of this study was to evaluate the association between AFV at the time of presentation with early preterm prelabor rupture of membranes (PPROM) and severe neonatal respiratory morbidity and other adverse pregnancy outcomes. METHODS: This was a retrospective study of all women with a singleton pregnancy, admitted to a single tertiary referral center between 2004 and 2014, for expectant management of PPROM at 20 + 0 to 28 + 6 weeks' gestation. The primary exposure was AFV at presentation, classified according to sonographic maximum vertical pocket (MVP) as: normal AFV (> 2 cm), oligohydramnios (≤ 2 cm and > 1 cm) or severe oligohydramnios (≤ 1 cm). The primary outcome was a composite variable of severe respiratory morbidity, defined as either of the following: (1) need for respiratory support in the form of mechanical ventilation using an endotracheal tube for ≥ 72 h and need for surfactant; or (2) bronchopulmonary dysplasia, defined as requirement for oxygen at postmenstrual age of 36 weeks or at the time of transfer to a Level-II facility. Adjusted odds ratios (aOR) and 95% CI for the primary and secondary outcomes were calculated for each AFV-at-presentation group (using normal AFV as the reference), adjusting for gestational age (GA) at PPROM, latency period, birth weight, mode of delivery and chorioamnionitis. RESULTS: In total, 580 women were included, of whom 304 (52.4%) had normal AFV, 161 (27.8%) had oligohydramnios and 115 (19.8%) had severe oligohydramnios at presentation. The rates of severe respiratory morbidity were 16.1%, 26.7% and 45.2%, respectively. Compared with normal AFV at presentation, oligohydramnios (aOR, 3.27; 95% CI, 1.84-5.84) and severe oligohydramnios (aOR, 4.11; 95% CI, 2.26-7.56) at presentation were associated independently with severe respiratory morbidity. Other variables that were associated independently with the primary outcome were GA at PPROM (aOR, 0.54; 95% CI, 0.43-0.69), latency period (aOR, 0.94; 95% CI, 0.91-0.98) and Cesarean delivery (aOR, 2.01; 95% CI, 1.21-3.32). CONCLUSIONS: In women with early PPROM, AFV at presentation, as assessed by the MVP on ultrasound examination, is associated independently with severe neonatal respiratory morbidity. This information may be taken into consideration when counseling women with early PPROM regarding neonatal outcome and management options. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Effect of intra-amniotic fluid pressure from polyhydramnios on cervical length in patients with twin-twin transfusion syndrome undergoing fetoscopic laser surgery.

OBJECTIVES: To determine the relationship between intra-amniotic pressure and cervical length (CL) in patients with twin-twin transfusion syndrome (TTTS) undergoing fetoscopic laser photocoagulation (FLP), and to identify pre- or intraoperative factors associated with increased intra-amniotic pressure in this population. METHODS: This was a prospective cohort study of patients undergoing FLP for TTTS. Exclusion criteria were triplet or higher-order gestation and prior cervical cerclage, amnioreduction or FLP procedure. CL was assessed using preprocedure transvaginal ultrasound. Intra-amniotic pressure measurements were obtained on initial placement of the trocar into the amniotic cavity, using a direct hydrostatic pressure gauge. The relationship between intra-amniotic pressure and CL was assessed using multivariate linear regression analysis, including relevant preoperative and intraoperative variables. RESULTS: In total, 283 pregnancies met the inclusion criteria. Quintero stage of TTTS was I in 33 pregnancies, II in 88, III in 150 and IV in 12. Mean gestational age (GA) at FLP was 20.7 ± 3 weeks. Mean intra-amniotic pressure was 23.1 ± 9 mmHg. On unadjusted linear regression analysis, there was no significant association between intra-amniotic pressure and preoperative CL (P = 0.24) or GA at delivery (P = 0.22). On multivariate analysis, the factors associated significantly with intra-amniotic pressure were: number of prior term deliveries (P = 0.03), recipient maximum vertical pocket (P < 0.0001), Quintero stage IV (P = 0.01) and type of anesthesia (sedation vs general anesthesia; P = 0.01). CONCLUSION: In pregnancies with TTTS, intra-amniotic pressure is not associated with CL or GA at delivery. This novel finding suggests that cervical shortening in this population is not mechanically driven. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Potential effects and molecular mechanisms of melatonin on the dopaminergic neuronal differentiation of human amniotic fluid mesenchymal stem cells.

Melatonin, a highly lipophilic molecule secreted by the pineal gland in the brain, plays a role in various biological functions. Previous studies reported that melatonin exerts its effect on mesenchymal stem cell (MSC) survival and differentiation into osteogenic- and adipogenic-lineage. However, the effect of melatonin in neurogenic differentiation in amniotic fluid (AF)-MSCs remains to be explored, thus we investigated the potential role of melatonin on dopaminergic neuron differentiation in AF-MSCs. The results showed that various concentrations of melatonin did not affect cell viability and proliferative effects of AF-MSCs. Increases in the levels of neuronal protein marker (ßIII-tubulin) and dopaminergic neuronal markers (tyrosine hydroxylase, TH and NURR1), but decrease in the level of glial fibrillary acidic protein (GFAP), were observed in melatonin-treated AF-MSCs. Melatonin induced alteration in differential expression patterns of mesenchymal stem cell antigens by reducing CD29, CD45, CD73, CD90 and CD105, but no changing CD34 expressing cells. AF-MSCs were sequentially induced in neurobasal medium containing standard inducing cocktails (ST: bFGF, SHH, FGF8, BDNF), 1 µM melatonin, or a combination of ST and melatonin. The levels of TUJ1, TH, MAP2, NURR1 and dopamine transporter (DAT) were significantly increased in all treated groups when compared with control-untreated cells. Pretreated AF-MSCs with non-selective MT1/MT2 receptors antagonist, luzindole and selective MT2 receptor antagonist, 4-P-PDOT diminished melatonin-induced increase in dopaminergic neuronal markers and phosphorylated ERK but did not diminish increase in phosphorylated CaMKII by melatonin. Pretreatment with mitogen-activated protein kinase (MEK) inhibitor, PD98059 and CaMKII inhibitor, KN-93 were able to abolish increase in the levels of dopaminergic markers in melatonin-treated AF-MSCs. These findings suggest that melatonin promotes dopaminergic neuronal differentiation of AF-MSCs possibly via the induction in ERK and CaMKII pathways through melatonin receptor-dependent and -independent mechanisms, respectively.

Amniotic Mesenchymal Stem Cells Decrease Aß Deposition and Improve Memory in APP/PS1 Transgenic Mice.

Transplantation of human amniotic mesenchymal stem cells (hAM-MSCs) seems to be a promising strategy for the treatment of neurodegenerative disorders, including Alzheimer's disease (AD). However, the clinical therapeutic effects of hAM-MSCs and their mechanisms of action in AD remain to be determined. Here, we used amyloid precursor protein (APP) and presenilin1 (PS1) double-transgenic mice to evaluate the effects of hAM-MSC transplantation on AD-related neuropathology and cognitive dysfunction. We found that hAM-MSC transplantation into the hippocampus dramatically reduced amyloid-ß peptide (Aß) deposition and rescued spatial learning and memory deficits in APP/PS1 mice. Interestingly, these effects were associated with increasing in Aß-degrading factors, elevations in activated microglia, and the modulation of neuroinflammation. Furthermore, enhanced hippocampal neurogenesis in the subgranular zone (SGZ) of the dentate gyrus (DG) and enhanced synaptic plasticity following hAM-MSC treatment could be another important factor in reversing the cognitive decline in APP/PS1 mice. Instead, the mechanism underlying the improved cognition apparently involves a robust increase in hippocampal synaptic density and neurogenesis that is mediated by brain-derived neurotrophic factor (BDNF). In conclusion, our data suggest that hAM-MSCs may be a new and effective therapy for the treatment of AD.

Reverse Thermal Gel for In Utero Coverage of Spina Bifida Defects: An Innovative Bioengineering Alternative to Open Fetal Repair.

Current state-of-the-art management of open spina bifida defects entails an open fetal surgery approach associated with significant morbidities. In an attempt to reduce these risks and provide for an earlier minimally invasive repair, it is aimed to develop and characterize an innovative alternative using a unique reverse thermal gel. This study focuses on characterization of the physical and biological properties of the polymer and its in vivo applicability. Based on the knowledge and benchmarking, the "ideal" biomaterial should have the following characteristics: stability in amniotic fluid, limited permeability, biocompatibility, biologically functional, nontoxic, ability to support cellular functions, and in vivo applicability. The results demonstrate that the polymer possesses a unique ultrastructure, is stable in amniotic fluid, possesses limited yet predictable permeability, biocompatible with cells exposed in neural tube defects, is nontoxic, and can support cellular migration. These characteristics make it a potential novel alternative to open fetal repairs.

Evaluation of perinatal outcomes in pregnant women with preterm premature rupture of membranes / Avaliação dos resultados perinatais em gestantes com rotura prematura das membranas pré-termo

SUMMARY Objective: To determine the association between amniotic fluid index (AFI) and perinatal outcomes in preterm premature rupture of membranes (PPROM). Method: A retrospective cohort study was conducted between 2008 and 2012. 86 pregnant women were included, with a diagnosis of PPROM and gestational age from 24 to 35 weeks. Women who presented hypertensive disorders, diabetes, fetuses with birth defects and infection at admission were excluded. To determine the association between AFI and perinatal outcomes, chi-square and Fisher’s exact test were used if necessary, as well as risk ratio (RR) and 95% confidence intervals (95CI). Correlation between AFI and perinatal outcomes was determined by using simple linear regression, and AFI progression during pregnancy was analyzed by Z-test. Results: When comparing newborns presenting ultrasound with AFI<5cm and AFI>5cm, there was a higher frequency of perinatal mortality when the AFI was lower than 5 cm. However, when the oligohydramnios was diagnosed as severe (AFI<3cm), there was a higher frequency of Apgar scores less than seven at 1 minute, neonatal sepsis and early neonatal mortality compared to those presenting AFI>3cm. There was a positive correlation between AFI and gestational age at delivery, birth weight and Apgar scores at minutes 1 and 5. There was also a decrease in amniotic fluid volume with increased gestational age. Conclusion: The presence of severe oligohydramnios after PPROM contributed to a higher frequency of perinatal complications and death.

RESUMO Objetivo: determinar a associação do índice de líquido amniótico (ILA) com os resultados perinatais na rotura prematura das membranas pré-termo (RPMPT). Método: realizou-se um estudo de coorte retrospectivo, de 2008 a 2012. Foram incluídas 86 gestantes, com diagnóstico de RPMPT e idade gestacional entre a 24ª e 35ª semanas. Foram excluídas gestantes que apresentavam síndromes hipertensivas, diabetes, fetos com malformações fetais e infecção na admissão. Para determinar a associação entre ILA e desfechos perinatais, foram utilizados os testes qui-quadrado e exato de Fisher, quando pertinentes, além da razão de risco (RR) e seu intervalo de confiança a 95% (IC95%). A correlação entre ILA e desfechos perinatais foi determinada por regressão linear simples, e a evolução do ILA durante a gestação foi analisada pelo teste Z. Resultados: quando comparados os recém-nascidos que apresentavam ultrassonografia com ILA<5 cm e ILA>5 cm, observou-se maior frequência de mortalidade perinatal nos casos de ILA<5 cm. Quando o oligo-hidrâmnio, porém, era diagnosticado como grave (ILA<3 cm), observava-se maior frequência de escore de Apgar <7 no 1º minuto, sepse neonatal e mortalidade neonatal precoce em relação aos que apresentavam ILA>3 cm. Observou-se uma correlação positiva entre ILA e idade gestacional no parto, peso ao nascer e escore de Apgar no 1º e 5º minutos, além de diminuição do volume do líquido amniótico com o avançar da idade gestacional. Conclusão: a presença de oligo-hidrâmnio grave após a RPMPT contribuiu para uma maior frequência de complicações e mortalidade perinatal.

In-utero radiofrequency ablation in fetal piglets: Lessons learned.

INTRODUCTION: Radiofrequency ablation (RFA) is increasingly utilized in minimally invasive fetal intervention. However, the response of different fetal tissues to RFA is poorly characterized. We sought to determine the extent of RFA damage in a fetal environment. METHODS: 90Day gestation Yorkshire piglets (term 115days) were subjected to RFA of the chest and abdominal viscera under various temperatures and wattages. The extent of tissue damage was determined by NADPH diaphorase histochemistry. RESULTS: Tyne temperature was widely variable and displayed varying responses between lung and liver tissue. Tyne exposure to amniotic fluid resulted in an increase in amniotic fluid temperature. Collateral damage, even across the diaphragm, was readily seen, and ultrasonography did not always reflect this injury. CONCLUSIONS: Utilization of extracorporeal tynes heats fluid at a greater rate than solid tissue and reliance on temperature sensitive probes may result in overheating. The extent of injury may extend beyond damage observed by ultrasound examination and varies for different tissues. Additional studies on the use of devices that regulate tyne temperature are needed to define optimal conditions and better define the extent of adjacent tissue injury.

The pathway not taken: understanding 'omics data in the perinatal context.

OBJECTIVE: 'Omics analysis of large datasets has an increasingly important role in perinatal research, but understanding gene expression analyses in the fetal context remains a challenge. We compared the interpretation provided by a widely used systems biology resource (ingenuity pathway analysis [IPA]) with that from gene set enrichment analysis (GSEA) with functional annotation curated specifically for the fetus (Developmental FunctionaL Annotation at Tufts [DFLAT]). STUDY DESIGN: Using amniotic fluid supernatant transcriptome datasets previously produced by our group, we analyzed 3 different developmental perturbations: aneuploidy (Trisomy 21 [T21]), hemodynamic (twin-twin transfusion syndrome [TTTS]), and metabolic (maternal obesity) vs sex- and gestational age-matched control subjects. Differentially expressed probe sets were identified with the use of paired t-tests with the Benjamini-Hochberg correction for multiple testing (P < .05). Functional analyses were performed with IPA and GSEA/DFLAT. Outputs were compared for biologic relevance to the fetus. RESULTS: Compared with control subjects, there were 414 significantly dysregulated probe sets in T21 fetuses, 2226 in TTTS recipient twins, and 470 in fetuses of obese women. Each analytic output was unique but complementary. For T21, both IPA and GSEA/DFLAT identified dysregulation of brain, cardiovascular, and integumentary system development. For TTTS, both analytic tools identified dysregulation of cell growth/proliferation, immune and inflammatory signaling, brain, and cardiovascular development. For maternal obesity, both tools identified dysregulation of immune and inflammatory signaling, brain and musculoskeletal development, and cell death. GSEA/DFLAT identified substantially more dysregulated biologic functions in fetuses of obese women (1203 vs 151). For all 3 datasets, GSEA/DFLAT provided more comprehensive information about brain development. IPA consistently provided more detailed annotation about cell death. IPA produced many dysregulated terms that pertained to cancer (14 in T21, 109 in TTTS, 26 in maternal obesity); GSEA/DFLAT did not. CONCLUSION: Interpretation of the fetal amniotic fluid supernatant transcriptome depends on the analytic program, which suggests that >1 resource should be used. Within IPA, physiologic cellular proliferation in the fetus produced many "false positive" annotations that pertained to cancer, which reflects its bias toward adult diseases. This study supports the use of gene annotation resources with a developmental focus, such as DFLAT, for 'omics studies in perinatal medicine.

The effect of human amniotic fluid on mandibular distraction osteogenesis.

The aim of this study was to evaluate the effects of local administration of human amniotic fluid (HAF) on newly formed bone obtained by mandibular distraction osteogenesis (DO) with histomorphometry. A unilateral mandibular osteotomy at the left corpus was performed in 32 adult male rabbits. After a 5-day latency period, the left mandibles were lengthened by mandibular DO over 5 days, at a rate of 1mm/day, via a custom-made distractor. After the distraction, the rabbits were divided randomly into four groups: 0.3 ml HAF was injected into the distraction gap followed by 21 (group 1) or 45 (group 2) days of consolidation; or 0.3 ml normal saline (NS) was administered followed by 21 (group 3) or 45 (group 4) days of consolidation. Mandibles were removed at the end of the consolidation period and investigated histomorphometrically. The newly formed bone area (NFBA) and number of fibroblasts increased significantly in the HAF groups compared to the NS groups (NFBA: group 1 vs. group 3, P<0.05; group 2 vs. group 4, P<0.01; fibroblasts: group 1 vs. group 3, and group 2 vs. group 4, P<0.05), and also in both 45-day consolidation groups compared to the 21-day consolidation groups (NFBA: group 1 vs. group 2, and group 3 vs. group 4, P<0.001; fibroblasts: group 1 vs. group 2, and group 3 vs. group 4, P<0.01). Additionally, the numbers of osteoblasts and capillaries were increased significantly at 45 days of consolidation compared to 21 days in both the HAF and NS groups (osteoblasts: group 1 vs. group 2, P<0.01; group 3 vs. group 4, P<0.05; capillaries: group 1 vs. group 2, and group 3 vs. group 4, P<0.01). Histomorphometric analysis demonstrated that local HAF administration effectively accelerated bone formation. Thus, a HAF injection procedure could improve new bone formation around the bone in maxillofacial operations such as DO.

Amniotic fluid exerts a neurotrophic influence on fetal neurodevelopment via the ERK/GSK-3 pathway

BACKGROUND: The fetus is surrounded by the amniotic fluid (AF) contained by the amniotic sac of the pregnant female. The AF is directly conveyed to the fetus during pregnancy. Although AF has recently been reported as an untapped resource containing various substances, it remains unclear whether the AF could influence fetal neurodevelopment. RESULTS: We used AF that was extracted from embryos at 16 days in pregnant SD rat and exposed the AF to the neural cells derived from the embryos of same rat. We found that the treatment of AF to cortical neurons increased the phosphorylation in ERK1/2 that is necessary for fetal neurodevelopment, which was inhibited by the treatment of MEK inhibitors. Moreover, we found the subsequent inhibition of glycogen synthase kinase-3 (GSK-3), which is an important determinant of cell fate in neural cells. Indeed, AF increased the neural clustering of cortical neurons, which revealed that the clustered cells were proliferating neural progenitor cells. Accordingly, we confirmed the ability of AF to increase the neural progenitor cells through neurosphere formation. Furthermore, we showed that the ERK/GSK-3 pathway was involved in AF-mediated neurosphere enlargement. CONCLUSIONS: Although the placenta mainly supplies oxygenated blood, nutrient substances for fetal development, these findings further suggest that circulating-AF into the fetus could affect fetal neurodevelopment via MAP kinases-derived GSK-3 pathway during pregnancy. Moreover, we suggest that AF could be utilized as a valuable resource in the field of regenerative medicine.

Therapeutic effects of amniotic fluid-derived mesenchymal stromal cells on lung injury in rats with emphysema.

BACKGROUND: In chronic obstructive pulmonary disease (COPD), two major pathological changes that occur are the loss of alveolar structure and airspace enlargement. To treat COPD, it is crucial to repair damaged lung tissue and regenerate the lost alveoli. Type II alveolar epithelial cells (AECII) play a vital role in maintaining lung tissue repair, and amniotic fluid-derived mesenchymal stromal cells (AFMSCs) possess the characteristics of regular mesenchymal stromal cells. However, it remains untested whether transplantation of rat AFMSCs (rAFMSCs) might alleviate lung injury caused by emphysema by increasing the expression of surfactant protein (SP)A and SPC and inhibiting AECII apoptosis. METHODS: We analyzed the phenotypic characteristics, differentiation potential, and karyotype of rAFMSCs, which were isolated from pregnant Sprague-Dawley rats. Moreover, we examined the lung morphology and the expression levels of SPA and SPC in rats with emphysema after cigarette-smoke exposure and intratracheal lipopolysaccharide instillation and rAFMSC transplantation. The ability of rAFMSCs to differentiate was measured, and the apoptosis of AECII was evaluated. RESULTS: In rAFMSCs, the surface antigens CD29, CD44, CD73, CD90, CD105, and CD166 were expressed, but CD14, CD19, CD34, and CD45 were not detected; rAFMSCs also strongly expressed the mRNA of octamer-binding transcription factor 4, and the cells could be induced to differentiate into adipocytes and osteocytes. Furthermore, rAFMSC treatment up-regulated the levels of SPA, SPC, and thyroid transcription factor 1 and inhibited AECII apoptosis, and rAFMSCs appeared to be capable of differentiating into AECII-like cells. Lung injury caused by emphysema was alleviated after rAFMSC treatment. CONCLUSIONS: rAFMSCs might differentiate into AECII-like cells or induce local regeneration of the lung alveolar epithelium in vivo after transplantation and thus could be used in COPD treatment and lung regenerative therapy.

Effect of amniotic fluid on the in vitro culture of human corneal endothelial cells.

The present study was designed to evaluate the effects of human amniotic fluid (HAF) on the growth of human corneal endothelial cells (HCECs) and to establish an in vitro method for expanding HCECs. HCECs were cultured in DMEM-F12 supplemented with 20% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using either FBS- or HAF-containing media. Cell proliferation and cell death ELISA assays were performed to determine the effect of HAF on cell growth and viability. The identity of the cells cultured in 20% HAF was determined using immunocytochemistry (ICC) and real-time reverse transcription polymerase chain reaction (RT-PCR) techniques to evaluate the expression of factors that are characteristic of HCECs, including Ki-67, Vimentin, Na+/K+-ATPase and ZO-1. HCEC primary cultures were successfully established using 20% HAF-containing medium, and these cultures demonstrated rapid cell proliferation according to the cell proliferation and death ELISA assay results. The ICC and real time RT-PCR results indicated that there was a higher expression of Na+/K+-ATPase and ZO-1 in the 20% HAF cell cultures compared with the control (20% FBS) (P < 0.05). The 20% HAF-containing medium exhibited a greater stimulatory effect on HCEC growth and could represent a potential enriched supplement for HCEC regeneration studies.

A vascular and thrombotic model of gastroschisis.

A binary vascular/thrombotic pathogenesis for gastroschisis, a form of congenital bowel herniation, is proposed, where normal right umbilical vein involution creates a possible site for thrombosis adjacent to the umbilical ring. If thrombosis occurs, it weakens the area, explaining overwhelmingly right-sided lesions. The model arises from the existence of two groups of risk factors with different maternal age associations. Older mothers show a greater association with vascular factors (although this may actually represent a lack of any significant maternal age effect), consistent with associations of gastroschisis with congenital heart lesions and with amyoplasia. Alternatively, other predispositions, and especially decreased maternal age, the greatest known risk factor, associate with factors raising maternal estrogen, with evidence that estrogen in turn acts here as a predisposition to thrombosis. Absorption of thrombotic by-products from the amniotic fluid can explain the unusual amniocyte inclusions that are common with gastroschisis, while a role for estrogens suggests a connection between rising gastroschisis prevalence and increasing environmental contamination with estrogen disruptors. This model explains a variety of structural and epidemiological findings, and suggests that stratification of data based on binary effects may clarify associated risks and mechanisms. The model also shows that what is often referred to as vascular disruption may actually reflect alternative or additional factors instead, including thrombosis as a primary mechanism.

Amniotic fluid may act as a transporting pathway for signaling molecules and stem cells during the embryonic development of amniotes.

Amniotic fluid (AF) is formed at the very early stages of pregnancy, and is present throughout embryonic development of amniotes. It is well-known that AF provides a protective sac around the fetus that allows fetal movement and growth, and prevents mechanical and thermal shock. However, a growing body of evidence has shown that AF contains a number of proteins and peptides, including growth factors and cytokines, which potently affect cellular growth and proliferation. In addition, pluripotent stem cells have recently been identified in AF. Herein, this article reviews the biological properties of AF during embryonic development and speculates that AF may act as a transporting pathway for signaling molecules and stem cells during amniote embryonic development. Defining this novel function of AF is potentially significant for further understanding embryonic development and regenerative medicine, preventing genetic diseases, and developing therapeutic options for human malignancies.

Genetic modification of primate amniotic fluid-derived stem cells produces pancreatic progenitor cells in vitro.

Insulin therapy for type 1 diabetes does not prevent serious long-term complications including vascular disease, neuropathy, retinopathy and renal failure. Stem cells, including amniotic fluid-derived stem (AFS) cells - highly expansive, multipotent and nontumorigenic cells - could serve as an appropriate stem cell source for ß-cell differentiation. In the current study we tested whether nonhuman primate (nhp)AFS cells ectopically expressing key pancreatic transcription factors were capable of differentiating into a ß-cell-like cell phenotype in vitro. nhpAFS cells were obtained from Cynomolgus monkey amniotic fluid by immunomagnetic selection for a CD117 (c-kit)-positive population. RT-PCR for endodermal and pancreatic lineage-specific markers was performed on AFS cells after adenovirally transduced expression of PDX1, NGN3 and MAFA. Expression of MAFA was sufficient to induce insulin mRNA expression in nhpAFS cell lines, whereas a combination of MAFA, PDX1 and NGN3 further induced insulin expression, and also induced the expression of other important endocrine cell genes such as glucagon, NEUROD1, NKX2.2, ISL1 and PCSK2. Higher induction of these and other important pancreatic genes was achieved by growing the triply infected AFS cells in media supplemented with a combination of B27, betacellulin and nicotinamide, as well as culturing the cells on extracellular matrix-coated plates. The expression of pancreatic genes such as NEUROD1, glucagon and insulin progressively decreased with the decline of adenovirally expressed PDX1, NGN3 and MAFA. Together, these experiments suggest that forced expression of pancreatic transcription factors in primate AFS cells induces them towards the pancreatic lineage.

Amniotic fluid and placental membranes: unexpected sources of highly multipotent cells.

Gestational tissue such as the placenta, placental membranes, and amniotic fluid are usually discarded following birth. Recently, researchers have identified gestational tissue as an untapped source of stem cells that are highly multipotent and possess potent immunosuppressive properties. Placental mesenchymal stem cells (MSCs), human amnion epithelial cells (hAECs), and amniotic fluid-derived stem cells (AFSCs) have been shown to differentiate into various cell types including adipogenic, osteogenic, myogenic, endothelial, pulmonary, neurogenic, hepatogenic, cardiac, and pancreatic lineages. Their immunomodulatory properties suggest that gestational stem cells may have an important application in the treatment of various inflammatory diseases such as graft versus host and autoimmune diseases. In clinical and preclinical studies, gestational stem cells have shown efficacy in the treatment of Crohn disease, lung disease, diabetes, repair of bone defects, heart disease, kidney disease, neural degeneration, and blood disorders. Stem cells derived from the placenta, placental membranes, and amniotic fluid are a valuable resource for the field of regenerative medicine.