scholarly journals ID:2041 Animal Component Free Medium Retains the Stability of Hypervariable Region 1 (HV1) of Mitochondrial DNA of Human Full Term Amniotic Fluid Mesenchymal Stem Cells

2017 ◽  
Vol 4 (S) ◽  
pp. 72
Author(s):  
Rohayu Izanwati Mohd Rawi
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Amniotic Fluid ◽  
Hypervariable Region ◽  
Human Amniotic Fluid ◽  
Hypervariable Region 1 ◽  
Three Samples ◽  
The Stability ◽  
Animal Component

Therapeutic potential of human amniotic fluid (hAF) cells in treating human disease is very promising. Furthermore it provides an alternative in isolating stem cells from an accessible source, where the fluid is merely discarded, thus circumventing the ethical concerns. Mesenchymal stem cell (MSC) is one of the heterogenous cell populations in the amniotic fluid (AF), however, its role and potential for stem cell therapy are yet to be discovered. One of the criteria in establishing the clinical grade of MSCs is the ability of the cells to retain their genomic stability in vitro prior to bedside applications. The microenviroment niche of the culture medium used is important to generate the safe MSCs. As mitochondria play the vital role in metabolic task such as apoptosis and cell proliferation, therefore investigating mitochondria DNA (mtDNA) is essential to mark the stability of stem cell. In this study, we aimed to detect variations of mtDNA across the passage number of cultured human amniotic fluid MSCs (hAFMSCs), cultured in animal component free (ACF) medium, specialised for human MSCs. Polymorphisms of hypervariable region 1 (HV1) in the D-loop of mtDNA were investigated in a series of cultured hAFMSCs (passage 1 and 3) from three different samples (three biological replicates) as well as in their respective fresh samples (control). Extracted DNA samples were subjected to PCR to detect the sequence variations of HV1 (nt16024-16365). The sequences were then analysed using SeqScape v2.5 by comparing the sequences with The Cambridge Reference Sequence (rCRS) for human mtDNA. The common C-to-T transition at position 16223 (C16223T) for all three samples was identified. Another common base transition was also identified for the other two samples at position 16311 (T16311C). However, one of the samples showed more variants; a C-to-T transition at position 16290 (C16290T), an A-to-G at position 16317 (A16317G) and a G-to-A at position 16319 (G16319A). All variants were identified within the sequence of around 341bp nucleotides and their polymorphisms frequency was consistent for all three samples regardless of their passage numbers when compared to control. This pattern of polymorphisms demonstrates the HV1 mtDNA integrity of hAFMSCs cultured in ACF medium, hence highly suggesting the ability of ACF medium in retaining the stability of stem cells mtDNA in vitro. The ACF medium may serve as a good medium in culturing hAFMSCs that is safe and stable for basic research in stem cell and therapeutic applications.

scholarly journals Genetic and epigenetic modifications induced by chemotherapeutic drugs: human amniotic fluid stem cells as an in-vitro model

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Prabin Upadhyaya ◽  
Alessandra Di Serafino ◽  
Luca Sorino ◽  
Patrizia Ballerini ◽  
Marco Marchisio ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Amniotic Fluid ◽  
Real Time ◽  
In Vitro Model ◽  
Chemotherapeutic Agents ◽  
Human Amniotic Fluid ◽  
Amniotic Fluid Stem Cells ◽  
Vitro Model

Abstract Background Bleomycin, etoposide and cisplatin (BEP) are three chemotherapeutic agents widely used individually or in combination with each other or other chemotherapeutic agents in the treatment of various cancers. These chemotherapeutic agents are cytotoxic; hence, along with killing cancerous cells, they also damage stem cell pools in the body, which causes various negative effects on patients. The epigenetic changes due to the individual action of BEP on stem cells are largely unknown. Methods Human amniotic fluid stem cells (hAFSCs) were treated with our in-vitro standardized dosages of BEP individually, for seven days. The cells were harvested after the treatment and extraction of DNA and RNA were performed. Real-time PCR and flow cytometry were conducted for cell markers analysis. The global DNA methylation was quantified using 5mC specific kit and promoter and CpG methylation % through bisulfite conversion and pyrosequencing. Micro- RNAs (miRNAs) were quantified with real-time qPCR. Results The cytotoxic nature of BEP was observed even at low dosages throughout the experiment. We also investigated the change in the expression of various pluripotent and germline markers and found a significant change in the properties of the cells after the treatments. The methylation of DNA at global, promoter and individual CpG levels largely get fluctuated due to the BEP treatment. Several tested miRNAs showed differential expression. No positive correlation between mRNA and protein expression was observed for some markers. Conclusion Cytotoxic chemotherapeutic agents such as BEP were found to alter stem cell properties of hAFSCs. Different methylation profiles change dynamically, which may explain such changes in cellular properties. Data also suggests that the fate of hAFSCs after treatment may depend upon the interplay between the miRNAs. Finally, our results demonstrate that hAFSCs might prove to be a suitable in-vitro model of stem cells to predict genetic and epigenetic modification due to the action of various drugs.

scholarly journals Human Amniotic Fluid Mesenchymal Stem Cells from Second- and Third-Trimester Amniocentesis: Differentiation Potential, Molecular Signature, and Proteome Analysis

2015 ◽  
Vol 2015 ◽  
pp. 1-15 ◽  
Author(s):  
Jurate Savickiene ◽  
Grazina Treigyte ◽  
Sandra Baronaite ◽  
Giedre Valiuliene ◽  
Algirdas Kaupinis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Amniotic Fluid ◽  
Expression Patterns ◽  
Specific Gene ◽  
Specific Cell ◽  
Third Trimester ◽  
Human Amniotic Fluid ◽  
Differentiation Potential

Human amniotic fluid stem cells have become an attractive stem cell source for potential applications in regenerative medicine and tissue engineering. The aim of this study was to characterize amniotic fluid-derived mesenchymal stem cells (AF-MSCs) from second- and third-trimester of gestation. Using two-stage protocol, MSCs were successfully cultured and exhibited typical stem cell morphological, specific cell surface, and pluripotency markers characteristics. AF-MSCs differentiated into adipocytes, osteocytes, chondrocytes, myocytes, and neuronal cells, as determined by morphological changes, cell staining, and RT-qPCR showing the tissue-specific gene presence for differentiated cell lineages. Using SYNAPT G2 High Definition Mass Spectrometry technique approach, we performed for the first time the comparative proteomic analysis between undifferentiated AF-MSCs from late trimester of gestation and differentiated into myogenic, adipogenic, osteogenic, and neurogenic lineages. The analysis of the functional and expression patterns of 250 high abundance proteins selected from more than 1400 demonstrated the similar proteome of cultured and differentiated AF-MSCs but the unique changes in their expression profile during cell differentiation that may help the identification of key markers in differentiated cells. Our results provide evidence that human amniotic fluid of second- and third-trimester contains stem cells with multilineage potential and may be attractive source for clinical applications.

G-CSF and Erythropoietin Stability in Amniotic Fluid during Simulated in vitro Digestion Conditions

2002 ◽  
Vol 18 (6) ◽  
pp. 310-315 ◽  
Author(s):  
Darlene A Calhoun ◽  
Brooke E Richards ◽  
Jason A Gersting ◽  
Sandra E Sullivan ◽  
Robert D Christensen
Keyword(s):  
Amniotic Fluid ◽  
Preterm Neonate ◽  
In Vitro Digestion ◽  
Human Amniotic Fluid ◽  
Time Zero ◽  
Simulated Digestion ◽  
The Stability ◽  
Stimulating Factor ◽  
Degree Of Degradation

Objective: To determine the stability of granulocyte colony-stimulating factor (G-CSF) and erythropoietin (Epo) in human amniotic fluid and recombinant G-CSF (Neupogen) and Epo (Epogen) in simulated amniotic fluid to digestions at pH concentrations of 3.2, 4.5, and 5.8 to assess their bioavailability to the neonate. Design: A simulated amniotic fluid containing Neupogen and Epogen was subjected to in vitro conditions that mimicked preprandial and postprandial neonatal intestinal digestion. Human amniotic fluid was tested using identical digestion conditions as well as human amniotic fluid to which Epogen and Neupogen had been added. Main Outcome Measures: The percentages of G-CSF/Epo and Neupogen/Epogen remaining after 1 and 2 hours of simulated digestions were compared with those at time zero, and concentrations at 2 hours were compared with those at 1 hour and time zero. Results: In simulated amniotic fluid at pH 3.2, significant degradation of G-CSF was observed at 1 hour (p = 0.03). No differences were observed at 1 or 2 hours for either pH 4.5 (p = 0.30 and 0.11, respectively) or pH 5.8 (p = 0.20 and 0.49, respectively). Human amniotic fluid exhibited significant degradation pH 3.2 (p = 0.04) and pH 4.5 (p < 0.05) at 1 hour; no difference was noted at pH 5.8 at 1 hour (p = 0.34). When additional Neupogen was added to human amniotic fluid, significant degradation was observed at pH 3.2 (p < 0.05) and pH 4.5 (p = 0.03) at 1 hour; no difference was noted at 1 hour at pH 5.8 (p = 0.11). In simulated amniotic fluid at pH 3.2, significant degradation of Epo occurred at 1 hour (p < 0.05). There were no differences at 1 hour for pH 4.5 (p = 0.50) or pH 5.8 (p = 0.17). Human amniotic fluid exhibited significant degradation at pH 3.2 (p < 0.05) and pH 4.5 (p < 0.05) at 1 hour; no difference was noted at 1 hour at pH 5.8 (p = 0.34). When additional Epogen was added to human amniotic fluid, significant degradation was observed at pH 3.2 (p = 0.001) and pH 4.5 (p = 0.003); no difference was noted at 1 hour at pH 5.8 (p = 0.31). Conclusions: G-CSF/Epo in human amniotic fluid and Neupogen/Epogen in simulated amniotic fluid are preserved to varying degrees during simulated digestion conditions. The degree of degradation of both cytokines was time- and pH-dependent. Measurable quantities of G-CSF and Epo are biologically available when swallowed by the fetus or a preterm neonate.

291 ISOLATION AND CHARACTERIZATION OF MESENCHYMAL STEM CELLS DERIVED FROM HUMAN AMNIOTIC FLUID

2011 ◽  
Vol 23 (1) ◽  
pp. 243 ◽  
Author(s):  
S.-A. Choi ◽  
J.-H. Lee ◽  
K.-J. Kim ◽  
E.-Y. Kim ◽  
K.-S. Park ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Amniotic Fluid ◽  
Adult Stem Cells ◽  
Embryonic Stem ◽  
Stem Cell Marker ◽  
Second Trimester ◽  
Cell Marker ◽  
Human Amniotic Fluid ◽  
Amniotic Fluid Stem Cells

Adult stem cells have the capacity to differentiate into several different cell types, although their differentiation potential is limited compared with that of embryonic stem cells. Thus, adult stem cells are regarded as an exciting source for new cell therapies. Recent observations also indicate that stem cells derived from second-trimester amniocentesis are pluripotent – capable of differentiating into multiple lineages, including representatives of all 3 embryonic germ layers. In addition, amniotic fluid stem cells can be used in the generation of disease- or patient-specific stem cells, and amniotic fluid stem cells could be an ideal source for autologous cell replacement therapy in the later life of the fetus. The aim of the present study was to investigate isolation and characterisation of human amniotic fluid-derived mesenchymal stem cells (hAFS). We successfully isolated and characterised hAFS. Amniotic fluid samples were collected in the second trimester (median gestational age: 16 weeks, range: 15–17 weeks) for prenatal diagnosis. Specimens (2 mL) were centrifuged and incubated in low-glucose DMEM supplemented with 10% FBS, 25 ng of basic fibroblast growth factor, and 10 ng of epidermal growth factor at 37°C with 5% CO2. Human amniotic fluid cell (passage 6) expression of stem cell specific markers OCT-4, SOX2, Rex1, FGF4, and NANOG was confirmed by RT-PCR. Flow cytometric analysis showed that hAFS (passage 10) were positive for CD44, CD29, CD146, STRO1, and CD90 but negative for CD19. Immunocytochemical analysis of hAFS (passage 11) also showed the expression of OCT-4, SSEA-1, CD44, CD29, CD146, STRO1, and CD90, but hAFS were negative for CD19 and CD14. In conclusion, according to the previous studies on other mammalians, hAFS are an appropriate source of pluripotent stem cells. Here, we demonstrated that hAFS have a high expression of stem cell specific marker, including embryonic stem cell marker and mesenchymal stem cell marker. Therefore, amniotic fluid may be a suitable alternative source of multipotent stem cells.

scholarly journals Combination of Epigallocatechin Gallate and Sulforaphane Counteracts In Vitro Oxidative Stress and Delays Stemness Loss of Amniotic Fluid Stem Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-13 ◽  
Author(s):  
Pasquale Marrazzo ◽  
Cristina Angeloni ◽  
Michela Freschi ◽  
Antonello Lorenzini ◽  
Cecilia Prata ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stem Cells ◽  
Stem Cell ◽  
Amniotic Fluid ◽  
Osteogenic Differentiation ◽  
Epigallocatechin Gallate ◽  
The Body ◽  
Gene Markers ◽  
Amniotic Fluid Stem Cells

Amniotic fluid stem cells (AFSCs) are characterized in vivo by a unique niche guarantying their homeostatic role in the body. Maintaining the functionality of stem cells ex vivo for clinical applications requires a continuous improvement of cell culture conditions. Cellular redox status plays an important role in stem cell biology as long as reactive oxygen species (ROS) concentration is finely regulated and their adverse effects are excluded. The aim of this study was to investigate the protective effect of two antioxidants, sulforaphane (SF) and epigallocatechin gallate (EGCG), against in vitro oxidative stress due to hyperoxia and freeze-thawing cycles in AFSCs. Human AFSCs were isolated and characterized from healthy subjects. Assays of metabolic function and antioxidant activity were performed to investigate the effect of SF and EGCG cotreatment on AFSCs. Real-time PCR was used to investigate the effect of the cotreatment on pluripotency, senescence, osteogenic and adipogenic markers, and antioxidant enzymes. Alkaline phosphatase assays and Alizarin Red staining were used to confirm osteogenic differentiation. The cotreatment with SF and EGCG was effective in reducing ROS production, increasing GSH levels, and enhancing the endogenous antioxidant defences through the upregulation of glutathione reductase, NAD(P)H:quinone oxidoreductase-1, and thioredoxin reductase. Intriguingly, the cotreatment sustained the stemness state by upregulating pluripotency markers such as OCT4 and NANOG. Moreover, the cotreatment influenced senescence-associated gene markers in respect to untreated cells. The cotreatment upregulated osteogenic gene markers and promoted osteogenic differentiation in vitro. SF and EGCG can be used in combination in AFSC culture as a strategy to preserve stem cell functionality.

In vitro differentiation into insulin-producing β-cells of stem cells isolated from human amniotic fluid and dental pulp

2013 ◽  
Vol 45 (8) ◽  
pp. 669-676 ◽  
Author(s):  
Gianluca Carnevale ◽  
Massimo Riccio ◽  
Alessandra Pisciotta ◽  
Francesca Beretti ◽  
Tullia Maraldi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Amniotic Fluid ◽  
Dental Pulp ◽  
In Vitro Differentiation ◽  
Β Cells ◽  
Human Amniotic Fluid

scholarly journals Nuclear Nox4 Role in Stemness Power of Human Amniotic Fluid Stem Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Tullia Maraldi ◽  
Marianna Guida ◽  
Manuela Zavatti ◽  
Elisa Resca ◽  
Laura Bertoni ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Amniotic Fluid ◽  
Human Stem Cells ◽  
Human Amniotic Fluid ◽  
Differentiation Potential ◽  
Amniotic Fluid Stem Cells ◽  
Target Molecules ◽  
Cellular Compartments

Human amniotic fluid stem cells (AFSC) are an attractive source for cell therapy due to their multilineage differentiation potential and accessibility advantages. However the clinical application of human stem cells largely depends on their capacity to expandin vitro, since there is an extensive donor-to-donor heterogeneity. Reactive oxygen species (ROS) and cellular oxidative stress are involved in many physiological and pathophysiological processes of stem cells, including pluripotency, proliferation, differentiation, and stress resistance. The mode of action of ROS is also dependent on the localization of their target molecules. Thus, the modifications induced by ROS can be separated depending on the cellular compartments they affect. NAD(P)H oxidase family, particularly Nox4, has been known to produce ROS in the nucleus. In the present study we show that Nox4 nuclear expression (nNox4) depends on the donor and it correlates with the expression of transcription factors involved in stemness regulation, such as Oct4, SSEA-4, and Sox2. Moreover nNox4 is linked with the nuclear localization of redox sensitive transcription factors, as Nrf2 and NF-κB, and with the differentiation potential. Taken together, these results suggest that nNox4 regulation may have important effects in stem cell capability through modulation of transcription factors and DNA damage.

scholarly journals Comparative proteomic analysis of two distinct stem-cell populations from human amniotic fluid

2015 ◽  
Vol 11 (6) ◽  
pp. 1622-1632 ◽  
Author(s):  
Rita Romani ◽  
Francesca Fallarino ◽  
Irene Pirisinu ◽  
Mario Calvitti ◽  
Anna Caselli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Amniotic Fluid ◽  
Proteomic Analysis ◽  
Cell Populations ◽  
Human Amniotic Fluid ◽  
Comparative Proteomic Analysis ◽  
Stem Cell Populations

Characterization of two types of stem cells isolated from human amniotic fluid.

scholarly journals Human Amniotic Fluid Stem Cells Do Not Differentiate Into Dopamine Neurons In Vitro or After Transplantation In Vivo

2009 ◽  
Vol 18 (7) ◽  
pp. 1003-1012 ◽  
Author(s):  
Angela E. Donaldson ◽  
Jingli Cai ◽  
Ming Yang ◽  
Lorraine Iacovitti
Keyword(s):  
Stem Cells ◽  
Amniotic Fluid ◽  
Dopamine Neurons ◽  
Human Amniotic Fluid ◽  
Amniotic Fluid Stem Cells

scholarly journals Osteogenic Differentiation of Human Amniotic Fluid Mesenchymal Stem Cells Is Determined by Epigenetic Changes

2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
Monika Glemžaitė ◽  
Rūta Navakauskienė
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Amniotic Fluid ◽  
Osteogenic Differentiation ◽  
Epigenetic Changes ◽  
Human Amniotic Fluid ◽  
Polycomb Repressive Complex 2 ◽  
Cell Staining

Osteogenic differentiation of human amniotic fluid derived mesenchymal stem cells (AF-MSCs) has been widely studiedin vitroandin vivoas a potential tool for regenerative medicine and tissue engineering. While most of the studies analyze changes in transcriptional profile during differentiation to date there is not much information regarding epigenetic changes in AF-MSCs during differentiation. The aim of our study was to evaluate epigenetic changes during osteogenic differentiation of AF-MS cells. Isolated AF-MSCs were characterized morphologically and osteogenic differentiation was confirmed by cell staining and determining expression of alkaline phosphatase and osteopontin by RT-qPCR. Variation in gene expression levels of pluripotency markers and specific microRNAs were also evaluated. Analysis of epigenetic changes revealed that levels of chromatin modifying enzymes such as Polycomb repressive complex 2 (PRC2) proteins (EZH2 and SUZ12), DNMT1, HDAC1, and HDAC2 were reduced after osteogenic differentiation of AF-MSCs. We demonstrated that the level of specific histone markers keeping active state of chromatin (H3K4me3, H3K9Ac, and others) increased and markers of repressed state of chromatin (H3K27me3) decreased. Our results show that osteogenic differentiation of AF-MSCs is conducted by various epigenetic alterations resulting in global chromatin remodeling and provide insights for further epigenetic investigations in human AF-MSCs.

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