Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Summary

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

Abstract

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Introduction

Human implantation and the emergence of the early placenta are historically difficult to investigate and remain largely unknown because human tissues are inaccessible at this stage when pregnancy is clinically undetectable. Animal models are inadequate, as human placentation has its own unique features compared to other eutherian mammals. For example, human placenta invades deeply into the decidua with some trophoblast cells reaching at least the inner third of the uterine myometrium while other cells remodel the uterine spiral arteries. Even our closest evolutionary ancestors, the non-human primates, show differences in placental morphology and trophoblast interactio....

Protocol

All human embryos have been donated with consent for use in research. Protocols for extended human embryo culture have been approved by the Western Institutional Review Board (study no. 1179872) and follow international guidelines. Any use of human embryos must be reviewed by the appropriate ethics and governing bodies associated with the research institution using this protocol.

1. Preparation

1. Prepare media and recovery plates one day prior to embryo warming in a sterile laminar f.......

Discussion

The development of the protocol to culture human embryos through implantation has allowed scientists to explore a previously uncharted time in development^8,9. Here, we use an extended culture system to culture human embryos and study early trophoblast differentiation before the formation of villous placenta. The methods described here allow us to collect different TB sublineages for use in downstream single-cell analysis. This work allows the scientific community.......

Materials

Name	Company	Catalog Number	Comments
3130 or 3110 Forma Series II water-jacketed CO2 incubator	Thermo Fisher Scientific	13-998-078
35 mm Corning Primaria tissue culture dish	VWR	62406-038	Case of 500
5 mL snap cap tube	VWR	60819-295	Pack of 25
60 mm Corning Primaria tissue culture dish	VWR	25382-687	Case of 200
6-well dish	Agtech Inc.	D18	Pack of 1, 10, or 50
Acidic Tyrode's solution	Millipore Sigma	T1788	100 mL
Biotix 1250 µL pipette tips	VWR	76322-156	Pack of 960
Blast, blastocyst culture media	Origio	83060010	10 mL
Dilution Solution	Kitazato	VT802	1 x 4 mL
Disposable Borosilicate Glass Pasteur Pipets	Thermo Fisher Scientific	1367820D	5.75 in. (146mm); 720/Cs
Dulbecco's Phosphate Buffered Saline	Millipore Sigma	D8537
Embryo culture paraffin oil OvOil	Vitrolife	10029	100 mL
Eppendorf PCR tubes 0.2 mL	VWR	47730-598	Pack of 1,000
Eppendorf PCR tubes 0.5 mL	VWR	89130-980	Case of 500
Fibronectin from human plasma. Liquid .1% solution	Millipore Sigma	F0895	1 mg
Gilson 1 mL Pipetteman	Thermo Fisher Scientific	F123602G	1 Pipetteman 200-1000 µL
Gilson 20 µL Pipetteman	Thermo Fisher Scientific	F123602G	1 Pipetteman 2-20 µL
Gilson 200 µL Pipetteman	Thermo Fisher Scientific	F123602G	1 Pipetteman 50-200 µL
G-MOPS handling media	Vitrolife	10129	125 mL
Handling media	Origio	83100060	60 mL
Ibidi 8 well chambered coverslip	Ibidi	80826	15 slides per box
IVC1/IVC2	Cell Guidance Systems	M11-25/ M12-25	5-5mL aliquots
K System T47 Warming Plate	Cooper Surgical	23054
MilliporeSigma Millex Sterile Syringe Filters with Durapore PVFD Membrane	Fisher Scientific	SLGVR33RS	Pack of 50
Mouth pieces	IVF Store	MP-001-Y	100 pieces
Oosafe center well dish	Oosafe	OOPW-CW05-1	Case of 500
Quinn's Advantage SPS	Origio	ART-3010	12x 12 mL
Rubber latex tubing for mouth pieces	IVF Store	IVFS-NRL-B-5	5 ft.
Stereomicroscope	Nikon	SMZ1270
Stripper tips	Cooper Surgical	MXL3-275	20/pk 275 µm
Thawing Solution	Kitazato	VT802	2 x 4 mL
The Stripper Micropipetter	Cooper Surgical	MXL3-STR
TrypLE Express Enzyme (1X), no phenol red	Thermo Fisher Scientific	12604013	1 x 100 mL
Tween20	Millipore Sigma	P1379-25ML	25 mL bottle
VWR 1-20 µL pipette tips	VWR	76322-134	Pack of 960
VWR 1-200 µL pipette tips	VWR	89174-526	Pack of 960
Washing Solution	Kitazato	VT802	1 x 4 mL