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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.


Morphological characteristics of AF cells. (a) Amniotic fluid cells from amniocentesis sample. (b) The colony appearance of epithelial type at 10–15 days after initiation of the primary culture and the expansion of epithelial cell population at passage 3. (c, d) Mesenchymal-type cells in the primary culture at 10–15 days and after culturing to elongated spindle-shaped or flat “stromal” cell populations at passage 3.
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Related In: Results  -  Collection


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fig1: Morphological characteristics of AF cells. (a) Amniotic fluid cells from amniocentesis sample. (b) The colony appearance of epithelial type at 10–15 days after initiation of the primary culture and the expansion of epithelial cell population at passage 3. (c, d) Mesenchymal-type cells in the primary culture at 10–15 days and after culturing to elongated spindle-shaped or flat “stromal” cell populations at passage 3.

Mentions: As reported previously [9, 24], the amniotic fluid cells represent a heterogeneous population and only 1% of them demonstrate stem cell characteristics. In this study, a two-step cultivation protocol [22] was used for producing AF cell population with homogeneous fibroblast-like morphology. Adherent cells derived from fresh AF samples of late second- and third-trimester in primary culture resulted in a mixed population with spindle- and round-shaped morphology forming colonies at about 15–20 days of culture (Figure 1(a)). The colonies of small and spherical cells with a centrally located nucleus resemble epithelial cells that form islands in culture of 2-3 passages (Figure 1(b)). The primary culture contained a slowly growing cell population, which displayed large and flat “stromal” cells with irregular cytoplasmic extensions and very small nucleus at the edge of cytoplasm (Figure 1(d)). Spindle-shaped cells of fibroblast-like morphology with a high proliferation potential were predominantly present in culture after the second and third passage as well (Figure 1(c)). The derivation of mesenchymal stem cell population using two-step protocol was successful, and after 4–8 passages in culture, the population became morphologically homogeneous with fibroblastic morphology (Figures 1(c) and 2(a)). These AF-MSCs grew to 80–90% confluence of the subsequent passage culture in 2–4 days and were tested on their cellular phenotypic characteristics and differentiation potential at passages 4–6.


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)

Morphological characteristics of AF cells. (a) Amniotic fluid cells from amniocentesis sample. (b) The colony appearance of epithelial type at 10–15 days after initiation of the primary culture and the expansion of epithelial cell population at passage 3. (c, d) Mesenchymal-type cells in the primary culture at 10–15 days and after culturing to elongated spindle-shaped or flat “stromal” cell populations at passage 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Morphological characteristics of AF cells. (a) Amniotic fluid cells from amniocentesis sample. (b) The colony appearance of epithelial type at 10–15 days after initiation of the primary culture and the expansion of epithelial cell population at passage 3. (c, d) Mesenchymal-type cells in the primary culture at 10–15 days and after culturing to elongated spindle-shaped or flat “stromal” cell populations at passage 3.
Mentions: As reported previously [9, 24], the amniotic fluid cells represent a heterogeneous population and only 1% of them demonstrate stem cell characteristics. In this study, a two-step cultivation protocol [22] was used for producing AF cell population with homogeneous fibroblast-like morphology. Adherent cells derived from fresh AF samples of late second- and third-trimester in primary culture resulted in a mixed population with spindle- and round-shaped morphology forming colonies at about 15–20 days of culture (Figure 1(a)). The colonies of small and spherical cells with a centrally located nucleus resemble epithelial cells that form islands in culture of 2-3 passages (Figure 1(b)). The primary culture contained a slowly growing cell population, which displayed large and flat “stromal” cells with irregular cytoplasmic extensions and very small nucleus at the edge of cytoplasm (Figure 1(d)). Spindle-shaped cells of fibroblast-like morphology with a high proliferation potential were predominantly present in culture after the second and third passage as well (Figure 1(c)). The derivation of mesenchymal stem cell population using two-step protocol was successful, and after 4–8 passages in culture, the population became morphologically homogeneous with fibroblastic morphology (Figures 1(c) and 2(a)). These AF-MSCs grew to 80–90% confluence of the subsequent passage culture in 2–4 days and were tested on their cellular phenotypic characteristics and differentiation potential at passages 4–6.

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.