Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

Summary

This study presents a validated and optimized procedure for the isolation and culture of human endometrial stromal cells to conduct in vitro decidualization assay. Further, this study provides a detailed method to efficiently knockdown a specific gene using siRNAs in human endometrial stromal cells.

Abstract

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for embryo implantation into the uterine lining of the maternal womb. Improper decidualization has been established as a root cause for implantation failure and subsequent early embryo miscarriage. Therefore, understanding the molecular mechanisms underlying decidualization is advantageous to improving the rate of successful births. In vivo based studies of artificial decidualization are often limiting due to ethical dilemmas associated with human research, as well as translational complications within animal models. As a result, in vitro assays through primary cell culture are often utilized to explore the modulation of decidualization via hormones. This study provides a detailed protocol for the isolation of HESC and subsequent artificial decidualization via the supplementation of hormones to the culturing medium. Further, this study provides a well-designed method to knockdown any gene of interest by utilizing lipid-based siRNA transfections. This protocol permits the optimization of culture purity as well as product yield, thereby maximizing the ability to utilize this model as a reliable method to understand the molecular mechanisms underlying decidualization, and the subsequent quantification of secreted agents by decidualized endometrial stromal cells.

Introduction

Between the stages of menarche and menopause, women of reproductive age undergo monthly cycles of hormone-regulated endometrial proliferation, differentiation, and subsequent shedding in preparation for pregnancy in a process known as menstruation^1,2. Such physical modifications of the human endometrium are necessary for proper embryo implantation into the uterine wall¹. Alterations of the endometrium, including both morphological and biochemical adaptations, are mediated throughout the menstrual cycle via ovarian steroid hormones estrogen and progesterone (P4)³<....

Protocol

All human endometrium biopsies collected for this study were attained from the Washington University in St. Louis, Department of Obstetrics and Gynecology using an Institutional Review Board (IRB) approved written consent form.

1. Preparation

1. Prepare 500 mL of 1x Hank's Balanced Salt Solution (HBSS) by adding 100 U/mL penicillin and 100 µg/mL streptomycin to the media containing bottle (further referenced as HBSS+ medium).
2. Prepare 500 mL of Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) with phenol red, L-glutamine, and 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) by....

Results

Decidualization in HESC Culture

Following isolation, human endometrial stromal cells were cultured to a monolayer formation with 80-90% confluency and induced for in vitro decidualization by treating with 10 nM E2, 1 µM MPA, and 50 µM cAMP (EPC). Morphological shifts associated with in vitro decidualization were visualized in Figure 1A. Upon decidualization, HESCs .......

Discussion

The female reproductive menstrual cycle is characterized by a rise in progesterone levels throughout the luteal phase, thereby inducing the decidualization of ESC into round, epithelial-like secretory cells^3,8. The initiation of decidualization is species dependent. In humans, decidualization occurs spontaneously upon the rise of progesterone concentration, whereas mice require blastocyst presence^10,11. T.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Opti-MEM I (1X) Reduced Serum Medium	Gibco	31985-070	For decidualization media
DMEM / F12 (1:1) (1X)	Gibco	11330-032
Trypan Blue Stain (0.4%)	Gibco	15250-061	For cell count
PureLink RNA Mini Kit	Invitrogen	12183018A	For RNA Isolation/Purification
TaqMan 2X Universal PCR Master Mix	Applied Biosystems	4304437
High Capacity cDNA Reverse Transcription Kit	Applied Biosystems	4374967
Eukaryotic 18S rRNA Endogenous Control	Life Technologies	4319413E	For qPCR internal control
TaqMan Gene Expression Assay Prolactin Probe	Applied Biosystems	4331182	For qPCR internal control
TaqMan Gene Expression Assay IGFBP1 Probe	Applied Biosystems	4331182	For qPCR internal control
Phalloidin-iFluor 488 Reagent - CytoPainter	Abcam	ab176753
Human Prolactin ELISA Kit	Invitrogen	EHIAPRL
16% Paraformaldehyde	Alfa Aesar	30525-89-4	Fixative
Lipofectamine RNAiMAX	Invitrogen	13778150	Transfection Reagent
ProLong Gold antifade reagent with DAPI	Invitrogen	P36935	For mounting
0.25% Trypsin-EDTA (1X)	Gibco	25200-056
Penicillin Streptomycin	Gibco	15140-122
Charcoal Stripped Fetal Bovine Serum	Sigma	F6765-500ML
Sodium Bicarbonate (7.5%)	Gibco	25080-094
Ficoll-Paque PLUS Reagent	Fisher Scientific	45001749
Collagenase from Clostridium histolyticum	Sigma	C0130-1G
Deoxyribonuclease I from bovine pancreas	Sigma	DN25-100MG
Medroxyprogesterone 17-acetate	Sigma	M1629-1G	For EPC Media
Estradiol	Sigma	E1024-1G	For EPC Media
cAMP	Sigma	A6885-100MG	For EPC Media
Fine straight stitch scissors	Fine Science Tools	15396-00
TaqMan Gene Expression Assay NCOA2 Probe	Applied Biosystems	4351372
Fetal Bovine Serum, heat inactivated	Gibco	10-082-147
Antibiotic-antimycotic	Thermo Scientific	15240062
Hank’s Balanced Salt Solution	Corning	21-021-cv
50 mL conical polypropylene Falcon tube	Corning	352098
Dumont #5/45 Forceps	Fine Science Tools	11251-35
100 x 15 mm Glass Petri dish	VWR	75845-546
40 micron cell strainer	Midsci	229481
Isotemp 215 Water bath	Fisher Scientific	FS-215
LSE Centrifuge	Corning	6755
Human NCOA2 siRNA	Dharmacon	L-020159-00	Gene specific qPCR probe
Steri-cycle i160 CO2 Incubator	Thermo Scientific	51030301
NanoDrop 2000	Thermo Scientific	ND-2000	For RNA quantification
Phosphate Buffered Saline	Fisher Scientific	50146771
75 cm Canted Neck Cell Culture flask	Corning	430641U
6 well Non-Pyrogenic Cell Culture Plate	Corning	3506
Pipet Controller Ultra	Corning	4099
Photoshop	Adobe	19.0.1.334
25 cm Canted Neck Cell Culture flask	Corning	7200876
Triton X-100	Sigma	X100-1L
15 mL conical polypropylene Falcon tube	Corning	352099
10 mL Syringe	BD	302995
1300 Series A2 Biological Safety Hood	Thermo Scientific	1377
accuspin Micro17 centrifuge	Fisher Scientific	13100675
7500 Fast real time PCR system	Applied Biosystems	3052632
EVOS FL Immunofluorescence Microscope	Life Technologies	01414-155G-291
Leica Inverted Light Microscope	Leica	DMi1
Illrustrator	Adobe	22.0.1.253
Excel Spreadsheets	Microsoft	2016
Deckglaser 18 mm cover glasses	NeuVitro	GG-18
Reichert Bright-Line Hemacytometer	Sigma	Z359629-1EA
2-mercaptoethanol	Fisher Bioreagents	BP176-100	For RNA lysis buffer
1.2mL External Threaded Polypropylene Cryogenic Vial	Corning	430658	For freezing HESC cells
Dimethyl Sulfoxide (DMSO)	Sigma	D2650-100mL	For freezing media