In Vitro Culture of Epithelial Cells from Different Anatomical Regions of the Human Amniotic Membrane

Summary

This protocol describes the isolation of epithelial cells from different anatomical regions of the human amniotic membrane to determine their heterogeneity and functional properties for possible application in clinical and physiopathological models.

Abstract

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that the amniotic epithelium is a homogeneous layer. The human amnion can be divided into three anatomical regions: reflected, placental, and umbilical. Each region has different physiological roles, such as in pathological conditions. Here, we describe a protocol to dissect human amnion tissue in three sections and maintain it in vitro. In culture, cells derived from the reflected amnion displayed a cuboidal morphology, while cells from both placental and umbilical regions were squamous. Nonetheless, all the cells obtained have an epithelial phenotype, demonstrated by the immunodetection of E-cadherin. Thus, because the placental and reflected regions in situ differ in cellular components and molecular functions, it may be necessary for in vitro studies to consider these differences, because they could have physiological implications for the use of HAEC in biomedical research and the promising application of these cells in regenerative medicine.

Introduction

Human amniotic epithelium cells (HAEC) originate during the early stages of embryonic development, at around eight days postfertilization. They arise from a population of squamous epithelial cells of the epiblast that derive from the innermost layer of the amniotic membrane¹. Thus, HAEC are considered remnants of pluripotent cells from the epiblast that have the potential to differentiate into the three germ layers of the embryo². In the last decade, diverse research groups have developed methods to isolate these cells from the amniotic membrane at the term of gestation to characterize their pres....

Protocol

This protocol was approved by the ethical committee of Instituto Nacional de Perinatología in Mexico City (Registry number 212250-21041). All procedures performed in these studies were in accordance with the ethical standards of the Instituto Nacional de Perinatología, the Helsinki Declaration, and the guidelines set forth in the Ministry of Health’s Official Mexican Standard.

1. Preparation

1. Prepare a solution of 1x PBS with EDTA. To do so, add 500 μL of 0.5 M .......

Discussion

We implemented a new protocol to isolate HAEC from term membranes. It differs from previous reports in that each membrane was divided into its three anatomical regions prior to isolation to analyze cells from each one.

One of the most critical steps in the protocol is the washing of the membrane to remove all blood clots, because they can interfere with the activity of trypsin when separating the epithelial cells. Failure to carry out this step properly can lead to obtaining a primary culture .......

Materials

Name	Company	Catalog Number	Comments
Culture reagents
2-Mercaptoethanol	Thermo Fisher Scientific/Gibco	21985023	55 mM
Animal-Free Recombinant Human EGF	Peprotech	AF-100-15
Antibiotic-Antimycotic	Thermo Fisher Scientific/Gibco	15240062	100X
Dulbecco's Modified Eagle Medium	Thermo Fisher Scientific/Gibco	12430054	Supplemented with high glucose and HEPES
EDTA	Thermo Fisher Scientific/Ambion	AM9260G	0.5 M
Embryonic stem-cell FBS, qualified	Thermo Fisher Scientific/Gibco	10439024
Non-Essential Amino Acids	Thermo Fisher Scientific/Gibco	11140050	100X
Paraformaldehyde	any brand
Phosphate-Buffered Saline	Thermo Fisher Scientific/Gibco	10010023	1X
Saline solution (sodium chloride 0.9%)	any brand
Sodium Pyruvate	Thermo Fisher Scientific/Gibco	11360070	100 mM
Trypsin/EDTA 0.05%	Thermo Fisher Scientific/Gibco	25300054
Disposable material
100 µm Cell Strainer	Corning/Falcon	352360
100 mm TC-Treated Culture Dish	Corning	430167
24-well Clear TC-treated Multiple Well Plates	Corning/Costar	3526
6-well Clear TC-treated Multiple Well Plates	Corning/Costar	3516
Non-Pyrogenic Sterile Centrifuge Tube	any brand		with conical bottom
Non-Pyrogenic sterile tips of 1,000 µl, 200 µl and 10 µl.
Sterile cotton gauzes
Sterile serological pipettes of 5, 10 and 25 mL	any brand
Sterile surgical gloves	any brand
Equipment
Biological safety cabinet
Centrifuge
Micropipettes
Motorized Pipet Filler/Dispenser
Sterile beakers of 500 mL
Sterile plastic cutting board
Sterile scalpels, scissors, forceps, clamps
Sterile stainless steel container
Sterile tray
Tube Rotator	MaCSmix
Antibodies and Kits			Antibody ID
Anti-E-cadherin	BD Biosciences	610181	RRID:AB_3975
Anti-KI67	Santa Cruz	23900	RRID:AB_627859)
Anti-NANOG	Peprotech	500-P236	RRID:AB_1268274
Anti-OCT4	Abcam	ab19857	RRID:AB_44517
Anti-SOX2	Millipore	AB5603	RRID:AB_2286686
Anti-SSEA-4	Cell Signaling	4755	RRID:AB_1264259
Anti-TRA-1-60	Cell Signaling	4746	RRID:AB_2119059
Goat Anti-Mouse Alexa Fluor 488	Thermo Fisher Scientific	A-11029	RRID:AB_2534088
Goat Anti-Rabbit Alexa Fluor 568	Thermo Fisher Scientific	A-11036	RRID:AB_10563566
Tunel Assay Kit	Abcam	66110