Epigenetic alterations in amniotic fluid mesenchymal stem cells derived from normal and fetus‐affected gestations: A focus on myogenic and neural differentiations

Gasiūnienė, Monika; Zentelytė, Aistė; Treigytė, Gražina; Baronaitė, Sandra; Savickienė, Jūratė; Utkus, Algirdas; Navakauskienė, Rūta

doi:10.1002/cbin.11099

Cited by 9 publications

(9 citation statements)

References 67 publications

(84 reference statements)

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“…AF‐MSCs were differentiated into three lineages, such as adipogenic, osteogenic, and chondrogenic, to confirm their differentiation potential in vitro as described in . Adipogenic differentiation was induced using STEMPro adipogenic differentiation medium (Gibco, Thermo Fisher Scientific) for 12 days and then lipid droplets were stained using freshly diluted Oil Red O solution (3:2 ratio in distilled water).…”

Section: Methodsmentioning

confidence: 99%

Long‐term cultivation of human amniotic fluid stem cells: The impact on proliferative capacity and differentiation potential

Gasiūnienė

Valatkaitė

Navakauskienė

2020

J of Cellular Biochemistry

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Human amniotic fluid mesenchymal stem cells (AF-MSCs) are a valuable, easily obtainable alternative source of SCs for regenerative medicine. Usually, amounts of cells required for the translational purposes are large thus the goal of this study was to assess the potency of AF-MSCs to proliferate and differentiate during long-term cultivation in vitro. AF-MSCs were isolated from amniotic fluid of healthy women in the second trimester of pregnancy and cultivated in vitro. AF-MSCs were cultivated up to 42 passages and they still maintained pluripotency genes, such as OCT4, SOX2, and NANOG, expression at a similar level as in the initial passages as determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR).Fluorescence-activated cell sorting analysis demonstrated that the cell surface markers CD34 (negative), CD44, and CD105 (positive) expression was also stable, only the expression of SCs marker CD90 decreased during the cultivation. The morphology of AF-MSCs changed over passage, acridine orange/ethidium bromide staining revealed that more cells entered into apoptosis and the first signs of aging were detected only at late passages (later than p33) using SA-β-gal assay. Concomitantly, the differentiation potential towards cardiomyogenic lineage, induced with DNA methyltransferases inhibitors decitabine, zebularine, and RG108, was impaired when comparing AF-MSCs at p31/33 with p6. The expression of cardiomyocytes genes MYH6, TNNT2, DES together with ion channels genes of the heart (sodium, calcium, and potassium) decreased in p31/33 induced AF-MSCs. AF-MSCs have a great proliferative capacity and maintain most of the characteristics up to 33 passages; however, the cardiomyogenic differentiation capacity decreases to a certain extent during the long-term cultivation. These results provide useful insights for the potential use of AF-MSCs for biobanking and broad applications requiring high yield of cells or repeated infusions. Hence, it is vital to take into account the passage number of AF-MSCs, cultivated in culture, when utilizing them in vivo or in clinical experiments. K E Y W O R D S amniotic fluid, cardiac, cell differentiation, pregnancy, stem cells Abbreviations: AF-MSCs, amniotic fluid mesenchymal stem cells; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; MSCs, mesenchymal stem cells; PE, phycoerythrin.

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Section: Methodsmentioning

confidence: 99%

Long‐term cultivation of human amniotic fluid stem cells: The impact on proliferative capacity and differentiation potential

Gasiūnienė

Valatkaitė

Navakauskienė

2020

J of Cellular Biochemistry

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“…No aneuploidies were detected during clinical diagnostics in samples from both groups. hAFSCs {n = 3 samples from healthy gestations and n = 3 samples from pathological gestations concomitant with polyhydramnios ([Pat1: diagnosed with fetal gastric atony], [Pat2: diagnosed with fetal esophageal atresia], [Pat3: diagnosed with Treacher Collins syndrome])} were isolated using a two-stage protocol as described in Gasiūnienė et al (2019b) (protocol approved by the Vilnius Regional Biomedical Research Ethics Committee, No. 158200-18/7-1049-550, version No.…”

Section: Isolation Of Hafscsmentioning

confidence: 99%

“…It should also be emphasized that scientific data about hAFSCs derived from fetus-affected gestations are very limited. Our group previously showed that hAFSCs from normal and fetus-affected gestations had similar stem cell characteristics and potential to differentiate toward mesodermal (adipogenic, osteogenic, chondrogenic, and myogenic) as well as ectodermal (neurogenic) lineages (Gasiūnienė et al, 2019b;Zentelytė et al, 2020). However, no scientific data exist on the metabolic characteristics of these two types of hAFSCs, nor are there any data on the neurogenic and neurotrophic potential of hAFSCs obtained exclusively from polyhydramnios samples.…”

Section: Introductionmentioning

confidence: 99%

Metabolic Profile and Neurogenic Potential of Human Amniotic Fluid Stem Cells From Normal vs. Fetus-Affected Gestations

Valiulienė

Zentelytė

Beržanskytė

et al. 2021

Front. Cell Dev. Biol.

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Human amniotic fluid stem cells (hAFSCs) possess some characteristics with mesenchymal stem cells (MSCs) and embryonic stem cells and have a broader differentiation potential compared to MSCs derived from other sources. Although hAFSCs are widely researched, their analysis mainly involves stem cells (SCs) obtained from normal, fetus-unaffected gestations. However, in clinical settings, knowledge about hAFSCs from normal gestations could be poorly translational, as hAFSCs from healthy and fetus-diseased gestations may differ in their differentiation and metabolic potential. Therefore, a more thorough investigation of hAFSCs derived from pathological gestations would provide researchers with the knowledge about the general characteristics of these cells that could be valuable for further scientific investigations and possible future clinical applicability. The goal of this study was to look into the neurogenic and metabolic potential of hAFSCs derived from diseased fetuses, when gestations were concomitant with polyhydramnios and compare them to hAFSCs derived from normal fetuses. Results demonstrated that these cells are similar in gene expression levels of stemness markers (SOX2, NANOG, LIN28A, etc.). However, they differ in expression of CD13, CD73, CD90, and CD105, as flow cytometry analysis revealed higher expression in hAFSCs from unaffected gestations. Furthermore, hAFSCs from “Normal” and “Pathology” groups were different in oxidative phosphorylation rate, as well as level of ATP and reactive oxygen species production. Although the secretion of neurotrophic factors BDNF and VEGF was of comparable degree, as evaluated with enzyme-linked immunosorbent assay (ELISA) test, hAFSCs from normal gestations were found to be more prone to neurogenic differentiation, compared to hAFSCs from polyhydramnios. Furthermore, hAFSCs from polyhydramnios were distinguished by higher secretion of pro-inflammatory cytokine TNFα, which was significantly downregulated in differentiated cells. Overall, these observations show that hAFSCs from pathological gestations with polyhydramnios differ in metabolic and inflammatory status and also possess lower neurogenic potential compared to hAFSCs from normal gestations. Therefore, further in vitro and in vivo studies are necessary to dissect the potential of hAFSCs from polyhydramnios in stem cell-based therapies. Future studies should also search for strategies that could improve the characteristics of hAFSCs derived from diseased fetuses in order for those cells to be successfully applied for regenerative medicine purposes.

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“…2.4 | RNA and miRNA expression analysis AF-MSCs from normal and fetus-diseased gestation were cultivated up to passage 5, then RNA was isolated for genes and miRNA expression analysis by RT-qPCR as described earlier. 20 To normalize all experimental data, as reference genes GAPDH and RNU 48 were used.…”

Section: Flow Cytometrymentioning

confidence: 99%

Epigenetic regulation of amniotic fluid mesenchymal stem cell differentiation to the mesodermal lineages at normal and fetus‐diseased gestation

Zentelytė

Gasiūnienė

Treigytė

et al. 2019

J of Cellular Biochemistry

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Human mesenchymal stem cells isolated from amniotic fluid (AF‐MSCs) demonstrate the potency for self‐renewal and multidifferentiation, and can, therefore, be a potential alternative source of stem cells adapted for therapeutic purposes. The object of this study is to evaluate the efficacy of MSCs from AF when the pregnancy is normal or when the fetus is affected during pregnancy to differentiate into mesodermal lineage tissues and to elucidate epigenetic states responsible for terminal adipogenic and osteogenic differentiation. The morphology of AF‐MSCs from two cell sources and the expression of the cell surface‐specific (CD44, CD90, and CD105) markers and pluripotency (Oct4, Nanog, Sox2, and Rex1) genes were quite similar and underwent mesodermal lineage differentiation because this is shown by the typical cell morphology and of genes’ expression specific for adipogenic (peroxisome proliferator‐activated receptor‐ɣ, adiponectin) and osteoblastic (alkaline phosphatase, osteopontin, and osteocalcin) differentiation. Terminal lineage‐specific differentiation was related to differential expression of miR‐17, miR‐21, miR‐34a, and miR‐146a, decreased levels of acetylated H4 and H3K9, trimethylated H3K4 and H3K9, and the retention of H3K27me3 along with a reduction in the levels of HDAC1, DNMT1, and PRC1/2 proteins (BMI1/SUZ12). No significant distinction could be identified in the levels of expression of all epigenetic or pluripotency markers between undifferentiated MSCs isolated from AF of normal gestation and pregnancy where the fetus was damaged and between those differentiated toward adipocytes or osteoblasts. The expressional changes of those marks and microRNAs that occurred during terminal differentiation to mesodermal tissues indicate subtle epigenetic regulation in AF‐MSCs when the condition of the fetus is healthy normal or diseased. More detailed studies of epigenetic mechanisms may offer a better understanding of AF‐MSCs differentiation in fetus‐diseased conditions and their usage in an autologous therapeutic application and prenatal disease research.

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Epigenetic alterations in amniotic fluid mesenchymal stem cells derived from normal and fetus‐affected gestations: A focus on myogenic and neural differentiations

Cited by 9 publications

References 67 publications

Long‐term cultivation of human amniotic fluid stem cells: The impact on proliferative capacity and differentiation potential

Long‐term cultivation of human amniotic fluid stem cells: The impact on proliferative capacity and differentiation potential

Metabolic Profile and Neurogenic Potential of Human Amniotic Fluid Stem Cells From Normal vs. Fetus-Affected Gestations

Epigenetic regulation of amniotic fluid mesenchymal stem cell differentiation to the mesodermal lineages at normal and fetus‐diseased gestation

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