Isolation and Differentiation of Amniotic Membrane Stem Cells Into Keratinocytes

Abstract

The human amniotic membrane is a highly abundant and readily available tissue that may be useful for regenerative medicine and cell therapy. The amniotic membrane stem cells can differentiate into multiple cell lineages; they have low immunogenicity and anti-inflammatory functions. This research aims to examine the protocols for the isolation of human amniotic membrane stem cells, including their phenotypic characterization and in vitro potential for differentiation toward keratinocytes. Human placentas were obtained from selected cesarean-sectioned births. We isolated amniotic stem cells by trypsin and collagenase B digestion and centrifuged with Percoll. After monolayer expansion of adherent cells, the cells were characterized by immunocytology with octamer-binding transcription factor 4 and differentiated into keratinocytes by treating the cells with insulin, hydrocortisone, BMP-4, and vitamin C. Protocol for isolation of stem cells from amniotic membrane has high efficiency. Differentiation markers of stem cells into keratinocytes, such as vimentin, cytokeratin (CK) 14, and CK19, were determined by reverse transcription-polymerase chain reaction increase over time in culture. Stem cells isolated from the amniotic membrane can differentiate into keratinocytes. It has opened the prospect of using stem cells to regenerate skin and clinical applications.

Keywords: amniotic membrane; cytokeratin; differentiation; keratinocytes; stem cells.

Fig. 1.

(A) Proliferative capacity of amniotic stem cells. Cells were cultured at low density on micro-test culture plates for 1, 3, 7, 10, or 14 days. The number of cells was calculated by MTS methods. The principle of this method is the reduction of tetrazolium salts (products that absorb light at 490 nm) by living cells which will be proportional to their numbers. The data are expressed as the means ± SD from five independent experiments. (B) Amniotic stem cells expressed OCT-4. Electrophoresis image of polymerase chain reaction product of OCT-4. Three different samples of amniotic membrane stem cells were put into three lanes (lanes 1, 2, and 3). OCT-4, octamer-binding transcription factor 4.

Fig. 2.

Immunohistochemistry staining of amniotic membrane stem cells after 7 days of culture with biomarkers octamer-binding transcription factor 4 (OCT-4), vimentin, cytokeratin (CK)14, CK19. 4′,6-diamidino-2-phenylindole: staining nuclei. Merge: merged images between the cell immunochemical staining image and the staining nucleus.

Fig. 3.

mRNA expression: (A) CK14 and (B) CK19. mRNA expression increased after the cells were cultured with vitamins C and BMP-4. The mRNA results were calculated in comparison with the control group (arbitrary unit). Protein expression of (C) OCT-4 and (D) CK14. OCT-4 protein decreased, while CK14 protein increased gradually with culture time. Actin was used to reference the amount of protein added to each electrophoresis well. CK, cytokeratin; OCT-4, octamer-binding transcription factor 4.