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 EBs to a 15 mL conical tube and wait until the material sinks to the bottom of the tube.
3. Remove medium and fix EBs with a 4% paraformaldehyde (PFA) solution in PBS for 30 minutes in room temperature.
   Note (!): For in situ hybridization antigen recovery should be done in order to avoid cross link reaction with antibodies due to the use of PFA^6,7.
4. Remove PFA solution and wash with PBS for 5 min.
5. EBs should be placed in serial dilution of PBS-buffered sucrose solutions (10, 20 and 30%, in sequence) at 25-28°C, each solution must be replaced every 30 min. The material can then be stocked in the 30% sucrose solution at 4°C until the embedding step with OCT.

2. Tissue Embedding, Guidance and Freezing

6. Carefully collect the EBs from the tube. Discharge as much sucrose solution as possible.
   
   Note (!): It is important to wet the internal wall of the tip with sucrose solution before collecting the EBs. This procedure can avoid EB to be attached to the tip.
7. Place EBs in the mold and remove remaining sucrose solution with filter paper.
   
   Note (!): It is important not to fill the mold with EBs to avoid overlapping.
8. Fill mold with OCT slowly, taking care not to re-suspend EBs. After that, place all EB at the center of the mold and remove bubbles with the pipette.
9. Mildly agitate for 15 minutes.
10. Surround the mold with crushed dry ice to freeze the sample. OCT should be completely frozen. At this point blocks can be stored at -70°C, for at least one year.

3. Cryosectioning Technique

11. Remove the frozen block from -70°C and place on cryostat previously cooled at a temperature between -18 and -21 °C.
    
    Note (!): Temperatures outside this range may cause troubles like sections curling, melting or cracking.
12. Detach the OCT block from the mold and place it into the cryostat support by adding more OCT.
13. It is important to orient the block properly and align it parallel to the edge of the blade.
    
    Note(!): As EBs are smaller than other tissue samples, this step is extremely important to minimize loss of material.
14. OCT blocks should be sectioned superficially until the surface plane is obtained. Thin sections (10μm) of the tissue specimen must be taken and mounted onto glass slides previously coated with 200 mg/mL poly L lysine (10 μL per slide) ^9,10.
    
    Note(!): To avoid torned sections pay attention to any damages at the blade.
15. All sections should be air-dried for 1 hour at room temperature and then be used or stored at -70°C until use.

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.

Materials

Name	Type	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