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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 .......

Representative Results

HAEC were isolated from each of the three anatomical regions of the amniotic membrane and individually cultured in vitro. After 48 h of culture, cells with an epithelial phenotype adhered to the surface of the plate, although the media also contained cell debris and floating cells, which were removed once the medium was changed (Figure 3).

During the processing of primary culture (passage zero, P0), some complications could arise that can interfere with the experi.......

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 .......

Acknowledgements

Our research was supported by grants from Instituto Nacional de Perinatología de México (21041 and 21081) and CONACYT (A1-S-8450 and 252756). We thank Jessica González Norris and Lidia Yuriria Paredes Vivas for the technical support.

....

Materials

NameCompanyCatalog NumberComments
Culture reagents
2-MercaptoethanolThermo Fisher Scientific/Gibco2198502355 mM
Animal-Free Recombinant Human EGFPeprotechAF-100-15
Antibiotic-AntimycoticThermo Fisher Scientific/Gibco15240062100X
Dulbecco's Modified Eagle MediumThermo Fisher Scientific/Gibco12430054Supplemented with high glucose and HEPES
EDTAThermo Fisher Scientific/AmbionAM9260G0.5 M
Embryonic stem-cell FBS, qualifiedThermo Fisher Scientific/Gibco10439024
Non-Essential Amino AcidsThermo Fisher Scientific/Gibco11140050100X
Paraformaldehydeany brand
Phosphate-Buffered SalineThermo Fisher Scientific/Gibco100100231X
Saline solution (sodium chloride 0.9%)any brand
Sodium PyruvateThermo Fisher Scientific/Gibco11360070100 mM
Trypsin/EDTA 0.05%Thermo Fisher Scientific/Gibco25300054
Disposable material
100 µm Cell StrainerCorning/Falcon352360
100 mm TC-Treated Culture DishCorning430167
24-well Clear TC-treated Multiple Well PlatesCorning/Costar3526
6-well Clear TC-treated Multiple Well PlatesCorning/Costar3516
Non-Pyrogenic Sterile Centrifuge Tubeany brandwith 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 mLany brand
Sterile surgical glovesany 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 RotatorMaCSmix
Antibodies and KitsAntibody ID
Anti-E-cadherinBD Biosciences610181RRID:AB_3975
Anti-KI67Santa Cruz23900RRID:AB_627859)
Anti-NANOGPeprotech500-P236RRID:AB_1268274
Anti-OCT4Abcamab19857RRID:AB_44517
Anti-SOX2MilliporeAB5603RRID:AB_2286686
Anti-SSEA-4Cell Signaling4755RRID:AB_1264259
Anti-TRA-1-60Cell Signaling4746RRID:AB_2119059
Goat Anti-Mouse Alexa Fluor 488Thermo Fisher ScientificA-11029RRID:AB_2534088
Goat Anti-Rabbit Alexa Fluor 568Thermo Fisher ScientificA-11036RRID:AB_10563566
Tunel Assay KitAbcam66110

References

  1. Shahbazi, M. N., et al. Self-organization of the human embryo in the absence of maternal tissues. Nature Cell Biology. 18 (6), 700-708 (2016).
  2. Garcia-Lopez, G., et al. Human amniotic epithelium (HAE) as a pos....

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