Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies

Summary

Pluripotent stem cells growing in suspension differentiate into embryoid bodies (EBs). Here we demonstrate how to obtain high quality EB cryosections useful for studying cellular and molecular aspects of embryogenesis, while preserving their organization as aggregates.

Abstract

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos ¹. A crucial stage in the differentiation of ES cells is the formation of embryoid bodies (EBs) aggregates ^{2, 3}. EB formation is based on spontaneous aggregation when ES cells are cultured in non adherent plates. Three-dimensional EB recapitulates many aspects of early mammalian embryogenesis and differentiate into the three germ layers: ectoderm, mesoderm and endoderm ⁴.

Immunofluorescence and in situ hybridization are widely used techniques for the detection of target proteins and mRNA present in cells of a tissue section ^{5, 6, 7}. Here we present a simple technique to generate high quality cryosections of embryoid bodies. This approach relies on the spatial orientation of EB embedding in OCT followed by the cryosection technique. The resulting sections can be subjected to a wide variety of analytical procedures in order to characterize populations of cells containing certain proteins, RNA or DNA. In this sense, the preparation of EB cryosections (10μm) are essential tools for histology staining analysis (e.g. Hematoxilin and Eosin, DAPI), immunofluorescence (e.g. Oct4, nestin) or in situ hybridization. This technique can also help to understand aspects of embryogenesis with regards to the maintenance of the tri-dimensional spherical structure of EBs.

Protocol

1. Fixation and Cryopreservation

Pluripotent stem cells were cultured onto mouse embryonic fibroblasts (MEF) inactivated with mitomycin C and maintained in DMEM/F12 supplemented with 20% knockout serum replacement (KSR) and 8ng/ mL of fibroblast growth factor (FGF-2). In order to induce EB formation H9 cells were transferred to non-adherent dishes and cultured for 7 days, maintained in DMEM/F12 supplemented with 15% KSR ⁸.

Note (!): This technique can be used for EBs derived from any embryonic and induced pluripotent stem cell.

1. Collect the EBs from the culture dish with a pipette.
2. Transfer E....

Discussion

The method described here provides an easy-to-follow protocol to obtain PFA fixed thin cryostat sections of embryoid bodies useful for immunofluorescence and in situ hybridization assays. The resulting cryosections permit the study of cellular and molecular aspects of human embryonic stem cells differentiation, while preserving their structure and organization as aggregates.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
D (+) Sucrose	Reagent	Vetec	228
Paraformaldehyde	Reagent	Rieden-de Haën	16005
PBS solution	Reagent	LGC Biotecnology	13-30259.05
Poly-L-lysine hydrobromide	Reagent	Sigma-Aldrich	P2636	200mg/mL in water
Tissue-Tek O.C.T. Compound	Reagent	Sakura Finetek	P2636
Sucrose Solution	Reagent			10% Sucrose Solution in PBS w/v
Sucrose Solution	Reagent			20% Sucrose Solution in PBS w/v
Sucrose Solution	Reagent			30% Sucrose Solution in PBS w/v
Conic Tube	Tool	Techno Plastic Products	91015	15mL conic tube
Plate shaker	Tool	Biomixer
Cryostat	Tool	Leica Microsystems	CM 1850
Mold for OCT platform	Tool			Plastic mold plataform