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

Savickiene J, Treigyte G, Baronaite S, Valiuliene G, Kaupinis A, Valius M, Arlauskiene A, Navakauskiene R - Stem Cells Int (2015)

Bottom Line: Using two-stage protocol, MSCs were successfully cultured and exhibited typical stem cell morphological, specific cell surface, and pluripotency markers characteristics.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology, Vilnius University, LT-08662 Vilnius, Lithuania.

ABSTRACT
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.

No MeSH data available.


Differentiation potential of AF-MSCs. AF-MSCs were obtained from amniocentesis samples of second- and third-trimester at passages 5–8, cultured without differentiation supplements (a), or maintained in differentiation media. (b) Representative images of AF-MSCs after adipogenic treatment showing the accumulation of lipid vacuoles by Oil Red O staining; osteogenic treatment by Alizarin Red staining for calcium mineralization; neurogenic or myogenic treatment showing the presence of neuron-like cells or multinucleated cells by staining with crystal violet, respectively, and chondrogenic treatment showing glycosaminoglycan production in chondrogenic pellets by Alcian Blue staining. (c) Relative expression of adiponectin, myogenin, nestin, and osteopontin by RT-qPCR is presented as n-fold increase over untreated control. Data are presented as the mean ± S.N. (p < 0.05) for three independent experiments.
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fig3: Differentiation potential of AF-MSCs. AF-MSCs were obtained from amniocentesis samples of second- and third-trimester at passages 5–8, cultured without differentiation supplements (a), or maintained in differentiation media. (b) Representative images of AF-MSCs after adipogenic treatment showing the accumulation of lipid vacuoles by Oil Red O staining; osteogenic treatment by Alizarin Red staining for calcium mineralization; neurogenic or myogenic treatment showing the presence of neuron-like cells or multinucleated cells by staining with crystal violet, respectively, and chondrogenic treatment showing glycosaminoglycan production in chondrogenic pellets by Alcian Blue staining. (c) Relative expression of adiponectin, myogenin, nestin, and osteopontin by RT-qPCR is presented as n-fold increase over untreated control. Data are presented as the mean ± S.N. (p < 0.05) for three independent experiments.

Mentions: Furthermore, AF-MSCs obtained from amniocentesis samples of different gestational time were analysed for their capacity to differentiate toward adipogenic, osteogenic, chondrogenic, myogenic, and neurogenic lineages. All clones of AF-MSCs from amniocentesis samples of second- (16–19 week) and third-trimester (34 week) that grew in culture beyond 4 or 8 passages were able to differentiate in all lineages tested (Figure 3). Cells cultured under adipogenic condition for 12 days accumulated lipid vacuoles and exhibited intense staining with Oil Red O (Figure 3(b)). RT-qPCR analysis showed very high expression level (456-fold increase of control) of adipocytes marker adiponectin (Figure 3(c)). Similarly, after culturing with osteogenic medium for 12 days, most of cells exhibited extracellular matrix mineralization detected by Alizarin Red S staining. In this cell culture, the analysis of osteogenic transcript levels determined by RT-qPCR showed unexpected low expression of osteopontin relative to untreated control, expressing this gene too. At 6–12 days after neural induction, morphologically neural-like cells were observed by light microscope after staining with 0.1% crystal violet. In differentiated cells, the expression level of Nestin detected by RT-qPCR analysis was upregulated (28-fold increase) relative to untreated control, where Nestin is expressed as well. Myogenic differentiation was obvious by the presence of multinucleated cells as determined by phase contrast microscope after staining with 0.1% crystal violet (Figure 3(b)) and the expression of myogenin (12,3-fold increase) detected by RT-qPCR (Figure 3(c)). For the induction of chondrogenic differentiation, MSCs were cultured in high-density pellet mass culture. Chondrogenic differentiation was determined after 20 days by the appearance of chondrogenic pellet and the glycosaminoglycan production detected by Alcian Blue staining. Cultured AF-MSCs (control) did not show any of the above differentiation morphology (Figure 3(a)).


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

Savickiene J, Treigyte G, Baronaite S, Valiuliene G, Kaupinis A, Valius M, Arlauskiene A, Navakauskiene R - Stem Cells Int (2015)

Differentiation potential of AF-MSCs. AF-MSCs were obtained from amniocentesis samples of second- and third-trimester at passages 5–8, cultured without differentiation supplements (a), or maintained in differentiation media. (b) Representative images of AF-MSCs after adipogenic treatment showing the accumulation of lipid vacuoles by Oil Red O staining; osteogenic treatment by Alizarin Red staining for calcium mineralization; neurogenic or myogenic treatment showing the presence of neuron-like cells or multinucleated cells by staining with crystal violet, respectively, and chondrogenic treatment showing glycosaminoglycan production in chondrogenic pellets by Alcian Blue staining. (c) Relative expression of adiponectin, myogenin, nestin, and osteopontin by RT-qPCR is presented as n-fold increase over untreated control. Data are presented as the mean ± S.N. (p < 0.05) for three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4553339&req=5

fig3: Differentiation potential of AF-MSCs. AF-MSCs were obtained from amniocentesis samples of second- and third-trimester at passages 5–8, cultured without differentiation supplements (a), or maintained in differentiation media. (b) Representative images of AF-MSCs after adipogenic treatment showing the accumulation of lipid vacuoles by Oil Red O staining; osteogenic treatment by Alizarin Red staining for calcium mineralization; neurogenic or myogenic treatment showing the presence of neuron-like cells or multinucleated cells by staining with crystal violet, respectively, and chondrogenic treatment showing glycosaminoglycan production in chondrogenic pellets by Alcian Blue staining. (c) Relative expression of adiponectin, myogenin, nestin, and osteopontin by RT-qPCR is presented as n-fold increase over untreated control. Data are presented as the mean ± S.N. (p < 0.05) for three independent experiments.
Mentions: Furthermore, AF-MSCs obtained from amniocentesis samples of different gestational time were analysed for their capacity to differentiate toward adipogenic, osteogenic, chondrogenic, myogenic, and neurogenic lineages. All clones of AF-MSCs from amniocentesis samples of second- (16–19 week) and third-trimester (34 week) that grew in culture beyond 4 or 8 passages were able to differentiate in all lineages tested (Figure 3). Cells cultured under adipogenic condition for 12 days accumulated lipid vacuoles and exhibited intense staining with Oil Red O (Figure 3(b)). RT-qPCR analysis showed very high expression level (456-fold increase of control) of adipocytes marker adiponectin (Figure 3(c)). Similarly, after culturing with osteogenic medium for 12 days, most of cells exhibited extracellular matrix mineralization detected by Alizarin Red S staining. In this cell culture, the analysis of osteogenic transcript levels determined by RT-qPCR showed unexpected low expression of osteopontin relative to untreated control, expressing this gene too. At 6–12 days after neural induction, morphologically neural-like cells were observed by light microscope after staining with 0.1% crystal violet. In differentiated cells, the expression level of Nestin detected by RT-qPCR analysis was upregulated (28-fold increase) relative to untreated control, where Nestin is expressed as well. Myogenic differentiation was obvious by the presence of multinucleated cells as determined by phase contrast microscope after staining with 0.1% crystal violet (Figure 3(b)) and the expression of myogenin (12,3-fold increase) detected by RT-qPCR (Figure 3(c)). For the induction of chondrogenic differentiation, MSCs were cultured in high-density pellet mass culture. Chondrogenic differentiation was determined after 20 days by the appearance of chondrogenic pellet and the glycosaminoglycan production detected by Alcian Blue staining. Cultured AF-MSCs (control) did not show any of the above differentiation morphology (Figure 3(a)).

Bottom Line: Using two-stage protocol, MSCs were successfully cultured and exhibited typical stem cell morphological, specific cell surface, and pluripotency markers characteristics.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology, Vilnius University, LT-08662 Vilnius, Lithuania.

ABSTRACT
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.

No MeSH data available.