Human Amniotic Fluid Research Articles

Activity of p53 in human amniotic fluid stem cells increases their potentiality as a candidate for stem cell therapy.

The potential use of stem cells as a therapeutic treatment for many neurological disorders, such as stroke, has spiked an interest in their properties. Due to limitations of the present-day treatments, regenerative and protective therapies could prove very beneficial if a safe and effective treatment is identified. Using human amniotic fluid stem (hAFS) cells could theoretically provide both neuroprotective and regenerative properties to patients, and knowledge of p53's activity and function could be a key component in understanding the behavior and characteristics of these stem cells to harness their full potential. Many recent studies on p53 have provided new and valuable information that could give rise to new ideas for treatment options. More specifically, p53's activity inside hAFS cells lead them closer to becoming a potential therapeutic stem cell. Other neuroprotective treatments, such as hyperoxia and hypoxia sessions, are showing positive results. In combination, these data are helping to get closer to an effective treatment for neurological disorders.

Glycodelin-A stimulates the conversion of human peripheral blood CD16-CD56bright NK cell to a decidual NK cell-like phenotype.

Does glycodelin-A (GdA) induce conversion of human peripheral blood CD16-CD56bright natural killer (NK) cells to decidual NK (dNK) cells to facilitate placentation? GdA binds to blood CD16-CD56bright NK cells via its sialylated glycans and converts them to a dNK-like cells, which in turn regulate endothelial cell angiogenesis and trophoblast invasion via vascular endothelial growth factor (VEGF) and insulin-like growth factor-binding protein 1 (IGFBP-1) secretion, respectively. dNK cells are the most abundant leucocyte population in the decidua. These cells express CD16-CD56bright phenotype. Peripheral blood CD16-CD56bright NK cells and hematopoietic precursors have been suggested to be capable of differentiating towards dNK cells upon exposure to the decidual microenvironment. These cells regulate trophoblast invasion during spiral arteries remodelling and mediate homoeostasis and functions of the endothelial cells. GdA is an abundant glycoprotein in the human decidua with peak expression between the 6th and 12th week of gestation, suggesting a role in early pregnancy. Indeed, GdA interacts with and modulates functions and differentiation of trophoblast and immune cells in the human feto-maternal interface. Aberrant GdA expression during pregnancy is associated with unexplained infertility, pregnancy loss and pre-eclampsia. CD16+CD56dim, CD16-CD56bright and dNK cells were isolated from human peripheral blood and decidua tissue, respectively, by immuno-magnetic beads or fluorescence-activated cell sorting. Human extravillous trophoblasts were isolated from first trimester placental tissue after termination of pregnancy. Biological activities of the cells were studied after treatment with GdA at a physiological dose of 5 μg/mL. GdA was purified from human amniotic fluid by immuno-affinity chromatography. Expression of VEGF, CD9, CD49a, CD151 and CD158a in the cells were determined by flow cytometry. Angiogenic proteins in the spent media of NK cells were determined by cytokine array and ELISA. Blocking antibodies were used to study the functions of the identified angiogenic proteins. Endothelial cell angiogenesis was determined by tube formation and trans-well migration assays. Cell invasion and migration were determined by trans-well invasion/migration assay. Binding of normal and de-sialylated GdA, and expression of L-selectin and siglec-7 on the NK cells were analysed by flow cytometry. The association between GdA and L-selectin on NK cells was confirmed by immunoprecipitation. Extracellular signal-regulated protein kinases (ERK) activation was determined by Western blotting and functional assays. GdA treatment enhanced the expression of dNK cell markers CD9 and CD49a and the production of the functional dNK secretory product VEGF in the peripheral blood CD16-CD56bright NK cells. The spent media of GdA-treated CD16-CD56bright NK cells promoted tube formation of human umbilical vein endothelial cells and invasiveness of trophoblasts. These stimulatory effects were mediated by the stimulatory activities of GdA on an ERK-activation dependent production of VEGF and IGFBP-1 by the NK cells. GdA had a stronger binding affinity to the CD16-CD56bright NK cells as compared to the CD16+CD56dim NK cells. This GdA-NK cell interaction was reduced by de-sialylation. GdA interacted with L-selectin, expressed only in the CD16-CD56bright NK cells, but not in the CD16+CD56dim NK cells. Anti-L-selectin functional blocking antibody suppressed the binding and biological activities of GdA on the NK cells. N/A. Some of the above findings are based on a small sample size of peripheral blood CD16-CD56bright NK cells. These results need to be confirmed with human primary dNK cells. This is the first study on the biological role of GdA on conversion of CD16-CD56bright NK cells to dNK-like cells. Further investigation on the glycosylation and functions of GdA will enhance our understanding on human placentation and placenta-associated complications with altered NK cell biology. This work was supported by the Hong Kong Research Grant Council Grant 17122415, Sanming Project of Medicine in Shenzhen, the Finnish Cancer Foundation, Sigrid Jusélius Foundation and the Finnish Society of Clinical Chemistry. The authors have no competing interests to declare.

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 (βIII-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.

Human amniotic fluid stem cells have a unique potential to accelerate cutaneous wound healing with reduced fibrotic scarring like a fetus.

Adult wound healing can result in fibrotic scarring (FS) characterized by excess expression of myofibroblasts and increased type I/type III collagen expression. In contrast, fetal wound healing results in complete regeneration without FS, and the mechanism remains unclear. Amniotic fluid cells could contribute to scar-free wound healing, but the effects of human amniotic fluid cells are not well characterized. Here, we determined the effect of human amniotic fluid stem cells (hAFS) on FS during wound healing. Human amniotic fluid was obtained by amniocentesis at 15-17 weeks of gestation. CD117-positive cells were isolated and defined as hAFS. hAFS (1 × 106) suspended in PBS or cell-free PBS were injected around wounds created in the dorsal region of BALB/c mice. Wound size was macroscopically measured, and re-epithelialization in the epidermis, granulation tissue area in the dermis and collagen contents in the regenerated wound were histologically analyzed. The ability of hAFS to engraft in the wound was assessed by tracking hAFS labeled with PKH-26. hAFS fulfilled the minimal criteria for mesenchymal stem cells. hAFS injection into the wound accelerated wound closure via enhancement of re-epithelialization with less FS. The process was characterized by lower numbers of myofibroblasts and higher expression of type III collagen. Finally, transplanted hAFS were clearly observed in the dermis until day 7 implying that hAFS worked in a paracrine manner. hAFS can function in a paracrine manner to accelerate cutaneous wound healing, producing less FS, a process resembling fetal wound healing.

The immunomodulatory function of human amniotic fluid stromal cells on B lymphocytes

Current treatments for B cell-mediated disease are mainly based on global B cell depletion, thereby eliminating pathogenic B cells as well as Breg subsets. A more refined modulation of B cell activity could prove beneficial for patient treatment.Objective:To investigate the immunomodulatory function of human amniotic fluid stromal cells (hAFSCs) on different subpopulation of B lymphocytes.Methods:hAFSCs were isolated and cultured and identified by characteristic phenotypic markers. After coculture of B lymphocytes with hAFSCs, the activation, proliferation, differentiation, as well as apoptosis, cell cycle, and expression of the inhibitory costimulatory molecules B7H1, B7H3, and B7H4 of B lymphocytes were examined in vitro.Results:Coculture with hAFSCs significantly decreased the expression of CD80/CD86, Ki-67 and CFSE expression, on activated B lymphocytes. These might be due to the inhibition of B lymphocyte apoptosis and cell cycle arrest. In activated B lymphocytes, coculture with hAFSCs resulted in a reduced proportion of memory B and plasma cells, reduced amounts of immunoglobulins. hAFSCs could balance the B1 to B2 cell subpopulation ratio. hAFSCs could inhibit the expression of the negative co-inhibitory molecule B7H4 and PD-L1 on the activated B lymphocytes.Conclusion:hAFSCs could inhibit B cell activation, proliferation, and subpopulation differentiation. These might be due to their affect on B cell apoptosis, cell cycle and the expression of costimulatory molecules of human B lymphocytes. Our experiment provided the evidence for hAFSCs as ideal seed cells with therapeutic potential for treating humoral immunity disorders, which were mainly mediated by B lymphocytes.

Epigenetic and metabolic alterations in human amniotic fluid stem cells induced to cardiomyogenic differentiation by DNA methyltransferases and p53 inhibitors.

Human amniotic fluid-derived mesenchymal stem cells (AF-MSCs) may be a valuable source for cell therapy and regenerative medicine. In this study, the potential of DNA methyltransferases (DNMT) inhibitors Decitabine, Zebularine, RG108 alone or combined with Zebularine and p53 inhibitor Pifithrin-α to induce cardiomyogenic differentiation of AF-MSCs was investigated. Differentiation into cardiomyocyte-like cells initiation was indicated with all agents by changes in the cell phenotype, upregulation of the relative expression of the main cardiac genes (NKX2-5, TNNT2, MYH6, and DES) as well as of cardiac ion channels genes (sodium, calcium, and potassium) as determined by reverse-transcription quantitative polymerase chain reaction and the increase in Connexin43 levels as detected from Western blot and immunofluorescence data. Cellular energetics and mitochondrial function in induced cells were assessed using Seahorse analyzer and revealed the initiation of AF-MSCs metabolic transformation into cardiomyocyte-like cells. All used inducers were nontoxic to AF-MSCs, arrested cell cycle at the G0/G1 phase, and upregulated p53 and p21 expression. The relative expression of miR-34a and miR-145 that are related to cell cycle regulation was also observed. Furthermore, the evaluated levels of chromatin remodeling proteins enhancer of zeste homolog 2, suppressor of zeste 12 protein homolog, DNMT1, histone deacetylase 1 (HDAC1), HDAC2, and heterochromatin protein 1α, as well as the rate of activating histone modifications, exhibited rearrangements of chromatin after the induction of cardiomyogenic differentiation. In conclusion, we demonstrated that all explored DNMT and p53 inhibitors initiated cardiomyogenesis-related alterations in AF-MSCs through rather similar mechanisms but to a different extent providing useful insights for the future research and potential applications of AF-MSCs.

Quantifying steroid hormones in amniotic fluid by ultra-performance liquid chromatography and tandem mass spectrometry

Background: Simultaneous assessment of steroid hormone concentrations in amniotic fluid is of importance for elucidating long-term consequences of intrauterine processes, and of broad scientific and clinical relevance. The objective of the study was to develop sensitive and specific analytical ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) methods for determination of steroid hormone concentrations in human amniotic fluid, and to provide proof-of-concept of their applicability. Methods: Methods were validated according to linearity, limit-of-detection, limit-of-quantification, recovery, intra- and inter-assay precision, and applied to 275 amniotic fluid samples. Results: Limits-of-quantification (S/N=10:1) were 0.05 ng/mL for cortisol, dehydroepiandrosterone sulfate (DHEAS), estradiol, estriol, and testosterone, and 0.01 ng/mL and 1.0 ng/mL for cortisone and dehydroepiandrosterone (DHEA), respectively. Good inter- and intra-assay precision were observed. Cortisol, cortisone, DHEAS, estradiol and estriol concentration were quantified in all samples. By lack of sensitivity of the analytical method, DHEA was quantified in 11 samples only. Testosterone was quantified in 119 of 275 samples analyzed. 116 of the quantified testosterone samples were from male offspring (out of 138 male offspring). Conclusion: These specific and sensitive methods offer a simple and non-invasive way to measure cortisol, cortisone, DHEAS, estradiol, estriol, and testosterone concentrations in human amniotic fluid.

P53 Is Active in Human Amniotic Fluid Stem Cells.

Despite increasing interest in human amniotic fluid cells, very little is known about the regulation and function of p53 in this cell type. In this study, we show that undifferentiated human amniotic fluid cells express p53, yet at lower levels than in cancer cells. The p53 protein in amniotic fluid cells is mainly localized in the nuclei, however, its antiproliferative activity is compromised in these cells. Igf2, a maternal imprinted gene, and c-jun, a proto-oncogene, are regulated by p53 in these cells. DNA damage leads to an increase in p53 abundance in human amniotic fluid cells and to transcriptional activation of its target genes. Interestingly, cell differentiation toward the neural lineage leads to p53 induction as differentiation progresses.

The relationship between Aquaporin-2 protein levels in amniotic fluid and the fetal kidney in humans

ObjectivesAquaporin-2 (AQP2) is an important water channel protein that is expressed in the renal collecting duct and plays a key role in urine concentration and body water homeostasis. It has been demonstrated that the urinary excretion of AQP2 correlates strongly with its expression in the kidney in adult humans and rats. However, there have been no studies on the urinary excretion of AQP2 in human fetuses during development. Fetal urine is the main source of the amniotic fluid; we speculate that the level of AQP2 in the amniotic fluid could reflect the expression level of the AQP2 protein in the fetal kidney. The purpose of the present study was to explore the relationship between AQP2 in the amniotic fluid and that in the fetal kidney. MethodsIn the present study, the concentration of the AQP2 protein in human amniotic fluid was measured by Enzyme-linked immunosorbent assay (ELISA) and its expression level in human fetal kidneys were examined by wastern blot and immunohistochemistry. ResultsBoth the expression level of AQP2 in the fetal kidney (F = 195.9, P < 0.001) and the concentration of AQP2 in the amniotic fluid increased with gestational age (F = 1098, P < 0.001). Moreover, the concentration of AQP2 in the amniotic fluid was positively correlated with its expression level in the fetal kidney (r = 0.872, P < 0.0001). ConclusionsOur research indicates that AQP2 levels in the amniotic fluid may be used as a marker for AQP2 expression in the fetal kidney.

Stem cells transplantation positively modulates the heart-kidney cross talk in cardiorenal syndrome type II

IntroductionWe investigated the effects of human amniotic fluid stem cells (hAFS) and rat adipose tissue stromal vascular fraction GFP-positive cells (rSVC-GFP) in a model of cardio-renal syndrome type II (CRSII). Methods and resultsRHF was induced by monocrotaline (MCT) in 28 Sprague-Dawley rats. Three weeks later, four million hAFS or rSVC-GFP cells were injected via tail vein. BNP, sCreatinine, kidney and heart NGAL and MMP9, sCytokines, kidney and heart apoptosis and cells (Cs) engraftment were evaluated.Cell-treated rats showed a significant reduction of serum NGAL and Creatinine compared to CRSII. In both hAFS and rSVC-GFP group, kidney protein expression of NGAL was significantly lower than in CRSII (hAFS p = 0.036 and rSVC-GFP p < 0.0001) and similar to that of controls. In both hAFS and rSVC-GFP treated rats, we observed cell engraftment in the medulla and differentiation into tubular, endothelial and SMCs cells. Apoptosis was significantly decreased in cell-treated rats (hAFS 14.07 ± 1.38 and rSVC-GFP 12.67 ± 2.96 cells/mm2) and similar to controls (9.85 ± 2.1 cell/mm2). TUNEL-positive cells were mainly located in the kidney medulla. Pro-inflammatory cytokines were down regulated in cell-treated groups and similar to controls. In cell-treated rats, kidney and heart tissue NGAL was not complexed with MMP9 as in CRSII group, suggesting inhibition of MMPs activity. ConclusionCell therapy produced improvement in kidney function in rats with CRSII. This was the result of interstitial, vessel and tubular cell engraftment leading to tubular and vessel regeneration, decreased tubular cells apoptosis and mitigated pro-inflammatory milieu. Reduction of NGLA-MMP9 complexes mainly due to decrease MMPs activity prevented further negative heart remodeling.

A comparison of clinically relevant sources of mesenchymal stem cell-derived exosomes: Bone marrow and amniotic fluid

Background/PurposeExosomes may constitute a more practical alternative to live cells in select stem cell-based therapies. We sought to compare exosomes from two mesenchymal stem cell (MSC) sources relevant to perinatal and pediatric diseases. MethodsExosomes were isolated by reagent-enhanced centrifugation from cell culture media of banked human bone marrow (bm) and amniotic fluid (af) MSCs after serum starvation. Characterization was by flow exometry for tetraspanin markers CD9, CD63, and CD81, transmission electron microscopy for size and morphology, and tunable resistive pulse sensing for size distribution and concentration. Statistical comparisons of count data were made by Poisson regression modeling and Student's T-test. ResultsExosomes of appropriate size and morphology were isolated with comparable expressions of CD9 (96% vs. 94%), CD63 (88% vs. 66%), and CD81 (71% vs. 63%) for bmMSC and afMSC, respectively. Total exosome yield (particles/mL) adjusted for number of cells was higher from afMSCs than bmMSCs by an estimated 25% (P < 0.001). ConclusionsWhile bone marrow and amniotic fluid mesenchymal stem cells are comparable sources of exosomes in size distribution, morphology, and expression of typical surface markers, yield may be higher from amniotic fluid cells. The amniotic fluid appears to be a preferable source of exosomes for clinical applications. Level of evidenceN/A (bench laboratory study).

Assessment of 22 inorganic elements in human amniotic fluid: a cross-sectional study conducted in Canary Islands (Spain)

ABSTRACTWe conducted a cross-sectional study in the Hospital Universitario de Canarias (Tenerife, Canary Islands, Spain) measuring 22 inorganic elements in amniotic fluid (AF) samples obtained from 65 pregnant women. ICP-MS was used for quantification of inorganic elements. Newborn parameters at delivery were all within the normal range. Concentrations of all elements were detected in measurable amounts in AF. The concentration of elements was similar to those reported in the literature. The concentrations of the most dangerous heavy metals – Cd, Cr, Ni, Hg and Pb – were lower than those reported by other authors. This study demonstrates that toxic inorganic elements pass into and accumulate in AF. The presence of such pollutants in contact with developing embryos from the intrauterine period could exert adverse health effects that deserve future investigations.

Significance and expression of aquaporin-2 in fetus kidney and amniotic fluid at different stages of the pregnancy

Objective To explore the expression of aquaporin-2(AQP-2) in human fetus kidney and amniotic fluid at different stages of pregnancy. Methods Twenty-two cases of aborted fetuses′ kidneys were collected.They were divided into 3 groups according to the pregnancy age: 8 cases in 17-23+ 6 weeks, 8 cases in 24-31+ 6 weeks, and 6 cases in 32-38+ 6 weeks.Western blot was used to examine the expression of AQP-2 in the kidney.Twenty-four cases of the amniotic fluid were collected, and they were divided into 3 groups according to the pregnancy age: 10 cases in 17-23+ 6 weeks, 6 cases in 24-31+ 6 weeks, and 8 cases in 32-38+ 6 weeks.Eight cases of healthy adult morning urine were collected as positive controls.The AQP-2 protein in the amniotic fluid was detected with the method of enzyme-linked immunosorbent assay (ELISA) and the osmotic pressure of amniotic fluid at different stages of the pregnancy was measured with the freezing point osmometer. Results The expression of AQP-2 was increased with the extending of pregnancy age, and the AQP-2 expressions in fetus kidney of 17-23+ 6 weeks, 24-31+ 6 weeks and 32-38+ 6 weeks were 0.986±0.335, 1.566±0.272, and 2.080±0.246, respectively, and the difference was significant(P<0.05). The AQP-2 detected from amniotic fluid was positively correlated with the result of AQP-2 in the kidney(r=0.985, P<0.05), and the AQP-2 expression also increased with the extending of pregnancy age: 17-23+ 6 weeks, 24-31+ 6 weeks, 32-38+ 6 weeks and adult urine was (30.253±5.843) mg/L, (35.103±7.271) mg/L, and (42.580±1.230) mg/L and (46.493±0.450) mg/L, respectively.The osmolality of the amniotic fluid of 17-23+ 6 weeks, 24-31+ 6 weeks, 32-38+ 6 weeks was (272.600±4.827) mmol/L, (252.00±15.360) mmol/L, and (261.750±5.560) mmol/L, respectively, and the difference was significant(P<0.05). Conclusions The AQP-2 expression in human fetus kidneys has good correlation with amniotic fluid, which indicates that the level of AQP-2 of the amniotic fluid may reflect the expression of AQP-2 in the fetus kidney. Key words: Aquaporin-2; Kidney; Amniotic fluid; Fetus

Cardiomyocytes Derived from Human CardiopoieticAmniotic Fluids

Human amniotic fluid (hAF) cells share characteristics of both embryonic and adult stem cells. They proliferate rapidly and can differentiate into cells of all embryonic germ layers but do not form teratomas. Embryoid-bodies obtained from hAF have cardiac differentiation potential, but terminal differentiation to cardiomyocytes (CMs) has not yet been described. Our purpose was to promote cardiac differentiation in hAFcells. Cells were exposed to inducing factors for up to 15 days. Only the subset of hAF cells expressing the multipotency markers SSEA4, OCT4 and CD90 (CardiopoieticAF cells) responded to the differentiation process by increasing the expression of the cardiac transcription factors Nkx2.5 and GATA4, sarcomeric proteins (cTnT, α-MHC, α-SA), Connexin43 and atrial and ventricular markers. Furthermore, differentiated cells were positive for the calcium pumps CACNA1C and SERCA2a, with approximately 30% of CardiopoieticAF-derived CM-like cells responding to caffeine or adrenergic stimulation. Some spontaneous rare beating foci were also observed. In conclusion, we demonstrated that CardiopoieticAF cells might differentiate toward the cardiac lineage giving rise to CM-like cells characterized by several cardiac-specific molecular, structural, and functional properties.

The neurorestorative effect of human amniotic fluid stem cells on the chronic phase of neonatal hypoxic-ischemic encephalopathy in mice.

Hypoxic-ischemic encephalopathy (HIE) remains a major cause of cerebral palsy. Increasing evidence has suggested that mesenchymal stem cells have a favorable effect on HIE. However, the efficacy of human amniotic fluid stem cells (hAFS) for HIE, especially in the chronic phase, remains unclear. The aim of this study was to determine the neurorestorative effect of hAFS on the chronic phase of HIE. hAFS were isolated from AF cells as CD117-positive cells. HI was induced in 9-day-old mice. Animals intranasally received hAFS or phosphate-buffered saline at 10 days post HI and were harvested for histological analysis after functional tests at 21 days post HI. We also implanted PKH26-positive hAFS to assess their migration to the brain. Finally, we determined gene expressions of trophic factors in hAFS co-cultured with HI brain extract. hAFS improved sensorimotor deficits in HIE by gray and white matter restoration and neuroinflammation reduction followed by migration to the lesion. Brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), hepatocyte growth factor (HGF), and stromal cell-derived factor-1 (SDF-1) gene expressions in hAFS were elevated when exposed to HI-induced brain extract. hAFS induced functional recovery by exerting neurorestorative effects in HIE mice, suggesting that intranasal administration of hAFS could be a novel treatment for HIE, especially in the chronic phase.

Osteoblastic differentiation potential of human amniotic fluid-derived mesenchymal stem cells in different culture conditions

Osteoporosis is a bone degenerative disease characterized by a decrease in bone strength and an alteration in the osseous micro-architecture causing an increase in the risk of fractures. These diseases usually happen in post-menopausal women and elderly men. The most common treatment involves anti-resorptive agent drugs. However, the inhibition of bone resorption alone is not adequate for recovery in patients at the severe stage of osteoporosis who already have a fracture. Therefore, the combination of utilizing osteoblast micro mimetic scaffold in cultivation with the stimulation of osteoblastic differentiations to regain bone formation is a treatment strategy of considerable interest. The aims of this current study are to investigate the osteoblastic differentiation potential of mesenchymal stem cells derived from human amniotic fluid and to compare the monolayer culture and scaffold culture conditions. The results showed the morphology of cells in human amniotic fluid as f-type, which is a typical cell shape of mesenchymal stem cells. In addition, the proliferation rate of cells in human amniotic fluid reached the highest peak after 14 days of culturing. After which time, the growth rate slowly decreased. Moreover, the positive expression of specific mesenchymal cell surface markers including CD44, CD73, CD90, and also HLA-ABC (MHC class I) were recorded. On the other hand, the negative expressions of the endothelial stem cells markers (CD31), the hematopoietic stem cells markers (CD34, 45), the amniotic stem cells markers (CD117), and also the HLA-DR (MHC class II) were also recorded. The expressions of osteoblastogenic related genes including OCN, COL1A1, and ALP were higher in the osteogenic-induced group when compared to the control group. Interestingly, the osteoblastogenic related gene expressions that occurred under scaffold culture conditions were superior to the monolayer culture conditions. Additionally, higher ALP activity and greater calcium deposition were recorded in the extracellular matrix in the osteogenic-induced group than in the culture in the scaffold group. In summary, the mesenchymal stem cells derived from human amniotic fluid can be induced to be differentiated into osteoblastic-like cells and can promote osteoblastic differentiation using the applied scaffold.

Optimal Stem Cell Transporting Conditions to Maintain Cell Viability and Characteristics.

The preservation of stem cell viability and characteristics during cell transport from the bench to patients can significantly affect the success of cell therapy. Factors such as suspending medium, time, temperature, cell density, and container type could be considered for transport conditions. To establish optimal conditions, human amniotic fluid stem cells' (AFSCs) viabilities were analyzed under different media {DMEM(H), DMEM/F-12, K-SFM, RPMI 1640, α-MEM, DMEM(L), PBS or saline}, temperature (4, 22 or 37°C), cell density (1 × 107 cells were suspended in 0.1, 0.5, 1.0 or 2.0mL of medium) and container type (plastic syringe or glass bottle). After establishing the transport conditions, stem cell characteristics of AFSCs were compared to freshly prepared cells. Cells transported in DMEM(H) showed relatively higher viability than other media. The optimized transport temperature was 4°C, and available transport time was within 12h. A lower cell density was associated with a better survival rate, and a syringe was selected as a transport container because of its clinical convenience. In compare of stem cell characteristics, the transported cells with established conditions showed similar potency as the freshly prepared cells. Our findings can provide a foundation to optimization of conditions for stem cell transport.

Nano-sized CA125 antigen glycocamouflage: Mucin - Extracellular vesicles alliance to watch?

Mucin 16 (MUC16) is a transmembrane type mucin and its released extracellular portion is designated as CA125 antigen. It is considered to be part of a supramolecular glycoprotein complex having a complicated epitope map and extreme structural heterogeneity.Starting from the initial transmembrane localization of MUC16/CA125 antigen and its alternative routes of release by shedding or putative secretion, CA125 antigen from human amniotic fluid soluble and extracellular vesicles (EVs)-containing fractions were characterized aiming at the possible glycosylation-associated mode of distribution as a factor contributing to the reported conflicting structural data.Ultracentrifugation, sucrose density gradient centrifugation, ion-exchange chromatography and TEM were used for analysis.The results indicated that the smeared abundantly glycosylated high molecular mass CA125-immunoreactive species, which follow the wheat germ agglutinin-binding pattern, were shared across amniotic fluid soluble and particulate fractions. A lower molecular mass glycoprotein-like CA125-immunoreactive species which follows the peanut agglutinin-binding pattern and was specifically associated with the EVs-enriched fraction was observed.CA125 presentation in the particulate amniotic fluid fraction was found to be shaped by a complex interactome partially involving lactose-sensitive galectin-3 binding. The MUC16 - EVs alliance as well as heterogeneous mucin/macromolecular complexes, at membranes or extracellularly, may represent cryptic pools of distinct CA125 species.

Paper-Based Surface-Enhanced Raman Spectroscopy for Diagnosing Prenatal Diseases in Women.

We report the development of a surface-enhanced Raman spectroscopy sensor chip by decorating gold nanoparticles (AuNPs) on ZnO nanorod (ZnO NR) arrays vertically grown on cellulose paper (C). We show that these chips can enhance the Raman signal by 1.25 × 107 with an excellent reproducibility of <6%. We show that we can measure trace amounts of human amniotic fluids of patients with subclinical intra-amniotic infection (IAI) and preterm delivery (PTD) using the chip in combination with a multivariate statistics-derived machine-learning-trained bioclassification method. We can detect the presence of prenatal diseases and identify the types of diseases from amniotic fluids with >92% clinical sensitivity and specificity. Our technology has the potential to be used for the early detection of prenatal diseases and can be adapted for point-of-care applications.

S1P promotes migration, differentiation and immune regulatory activity in amniotic-fluid–derived stem cells

Stem cells have high potential for cell therapy in regenerative medicine. We previously isolated stem cell types from human amniotic fluid, derived from prenatal amniocentesis. One type, characterized by a fast doubling time, was designated as fast human amniotic stem cells (fHASCs). These cells exhibited high differentiation potential and immunoregulatory properties. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that influences stem-cell pluripotency, differentiation, mobility, and regulates immune functions. In this study, we investigated the influence of S1P on fHASC migration, proliferation, differentiation and immune regulatory functions. We found that fHASC stimulation with S1P potentiated their migratory and proliferative activity in vitro. Notably, short fHASC exposure to S1P enhanced their differentiation towards multiple lineages, including adipocytes, osteocytes and endothelial cells, an effect that was associated with downregulation of the main transcription factors involved in the maintenance of a stem-cell undifferentiated state. A specific crosstalk between S1P and tumor growth factor β1 (TGF-β1) has recently been demonstrated. We found that fHASC exposure to S1P in combination with TGF-β1 promoted the expression of the immune regulatory pathway of indoleamine 2,3-dioxygenase 1 (IDO1). In addition, human peripheral blood mononuclear cells, co-cultured with fHASCs treated with S1P and TGF-β1, expanded regulatory T-cells, via a mechanism requiring IDO1. Overall, this study demonstrates that S1P potentiates several properties in fHASCs, an effect that may be critical for exploiting the therapeutic potential of fHASCs and might explain the specific effects of S1P on stem cells during pregnancy.