Growth factors and their receptors derived from human amniotic cells in vitro

In vitro studies have shown that amnion-produced growth factors participated in angiogenesis, re-epithelialization, and immunomodulation. The aim of our study was to investigate the growth factors and receptors produced by human amnion tissue and amniotic cells. Human amnions (hAM) were isolated, and amnion circles were dissected for in vitro analysis. Some amnion fragments were digested by the use of different methods to obtain two cell fractions, which were analysed for mesenchymal and epithelial cell markers. Amniotic circles and human amniotic cell fractions were cultured in a protein-free medium. Proteins secreted into the culture medium were analysed with a human growth factor antibody array. Conditioned culture media were added to human umbilical vein epithelial cells (HUVECs) to test for stimulation of migration (scratch test) and proliferation (Ki67 expression). Fraction 1 cells expressed both cytokeratin and mesenchymal cell markers which indicated that it was composed of a mixture of human amnion epithelial cells (hAECs) and mesenchymal stromal cells (hAMSCs). Fraction 2 cells mainly expressed cytokeratin and, therefore, were designed as hAECs. Secretion of proteins by the cultured cells increased with time. The hAM cultures secreted EGF-R, IGF, and IGFBP-2,-3 and -6; Cell Fraction 1 secreted NT-4, whereas Cell Fraction 2 secreted G-CSF, M-CSF, and PDGF. Conditioned media of hAM cultures stimulated HUVECs migration. We have showed for the first time that human amnions and amniotic cells secreted IGFBP-6, MCSF-R, PDGF-AB, FGF-6, IGFBP-4, NT-4, and VEGF-R3. We found that Cell Fraction 1, Cell Fraction 2, and the whole amnion secreted different proteins, possibly due to different proportions of amnion-derived cells and different cell-cell interactions. The hAM cell factors remained functional in vitro and induced intensified migration of HUVECs. The growth factors and receptors found in amnion or amniotic cell media might be used for regenerative medicine

Effect of hAM CCM on HUVECs migration assayed by scratch test.

<p>There are results of 160 measurements, 8 independent assays with 10 measurements for test and control each. Median values and (P25, P75) are shown (n = 8, p < 0.05). Detailed description of the assay is in Material and methods.</p

The example of real time migration assay of stimulated and controlled HUVECs directly from the X-Celligence system.

<p>The assay was repeated seven times with similar result during 25h observation at 37^°C. The difference between migration curves for cells in cultures with presence of hAM CCM and in control medium was significant. (p < 0.05).</p

Growth factors in hAM CCM.

<p>Forty-one growth factors were quantitated by antibody array and the obtained values were combined into following growth factor families: EGF family (EGF-2, HB-EGF, EGF-R); FGF family (bFGF, FGF-4, FGF-6, FGF-7); Hematopoietic factors HF (MCSF, MCSF-R, SCF, SCF-R); IGF family (IGF-1, IGF-2, IGF-1SR); IGFBP family (IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6); Neurotrophic factors NF (bNGF, GDNF, NT-3, NT-4); PDGF family (PDGF-AA, PDGF-AB, PDGF-BB, PDGF-Ra, PDGF-Rb); TGF family (TGF-α, TGF-β, TGF-β2, TGF-β3); Vasculogenic factors VF (PLGF, VEGF, VEGF-R3, VEGF-D, VEGF-R2); some growth factors are presented separately: AR; G-CSF; GM-CSF; HGF. Each growth factor fluorescence value (FV) was measured and calculated as described in Materials and methods. (n = 4) (p < 0.05).</p

Effect of hAM CCM on chemotaxy indeks of BM MNCs.

<p>The chemotaxy index (CI) after 2.5 h at 37^°C incubation time was calculated by dividing the number of cells in lower chamber by the number of cells added to the upper chamber counted at the start of the test. Median values and interquartile range (P25, P75) are shown (n = 12, p < 0.05).</p

Effect of hAM CCM on HUVECs expression of cell adhesion molecule-1 (CD31) marker.

<p>Significantly lower expression is observed in presence of hAM CCM. Median values and interquartile ranges (P25, P75) are shown (n = 3, p < 0.05).</p