Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Summary

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

Abstract

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Introduction

Murine embryonic stem cells (ESCs) are pluripotent, remaining undifferentiated and self-renewing in the presence of the cytokine Leukemia Inhibitory Factor (LIF)¹. Upon withdrawal of LIF they will spontaneously differentiate into 3-dimensional (3D) structures called embryoid bodies (EBs)². The 3D architecture allows for the development of the three germ layers ectoderm, endoderm, and mesoderm, which then later give rise to mature tissue types³. ESCs are an exceptional model for elucidating the molecular mechanisms of cellular differentiation, particularly the investigation of the development of early hematopoietic progenitor cells (HPC....

Protocol

1. Embryoid Body (EB) Formation

1. Gelatin-adapt ESCs that have been maintained on mouse embryo fibroblasts (MEFs). Passage ESCs 3 times on 6 well tissue culture plate coated with 0.1% gelatin to remove MEFs.
   1. Grow cells in ESC maintenance media with LIF to keep the cells undifferentiated (Table 1). Make sure cells never exceed 80% confluence. Use low passage (10 or less passages after removing from MEFs) cells for differentiation.
2. On the day before ESCs are cultured for EB differentiation, passage the cells so they will be approximately 50-70% confluent the next day. Continue culturing cells in ESC maintena....

Results

In these studies gelatinized RW4 (Derived from 129X1/SvJ mouse strain) ESCs were used. EBs were isolated at 3 days of differentiation. For best results the EBs should be spherical and non-adherent (Figure 1A, B). After spinoculation with virus co-expressing the fluorescent marker GFP along with a gene of interest, EBs were reformed by the hanging drop method. EBs were successfully reformed from cell suspensions prepared from 2.0, 2.5, and 3.0 day EBs. However, EBs could not be reformed from cell suspensi.......

Discussion

As discussed above, ESC clones that inducibly express a gene of interest can be generated using doxycycline systems, however, generation of these lines is time-consuming and labor intensive. Described in this protocol is a method to express a gene of interest in single cell suspension prepared from ESC derived EBs. These infected cells are then reformed into EBs by hanging drop to examine subsequent differentiation. In the example (Figure 3), it is shown that expression of the mirn23a cluster en.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Sigma/Aldrich	D5796
β-mercaptoethanol	Sigma/Aldrich	M3148
FBS (ES Screened)	Hyclone	SH30070.03E
FBS (Defined)	Hyclone	SH30070.03
Non-essential Amino Acids	Life Technologies	11140
L-Glutamine	Life Technologies	35050
Penn/Strep	Life Technologies	15070
Trypsin/EDTA	Life Technologies	25200
LIF ESGRO	Millipore	ESG1107
ACCUMAX	Millipore	SCR006
Trypsin/EDTA	Life Technologies	25200
Falcon Tissue Culture Plates 6-well	Fisher Scientific	08-772-1B
Falcon Non-treated Plates 6-well	Fisher Scientific	08-772-49
Falcon Petri dish 10 cm	Fisher Scientific	08-757-100D
Falcon Petri dish 15 cm	Fisher Scientific	08-757-148
Falcon Tube 5 ml	Fisher Scientific	14-959-11A
Falcon Tube 5 ml with Cell Strainer Cap	Fisher Scientific	08-771-23
White sterile resevoirs	U.S.A. Scientific	111-0700
Rat anti-mouse CD41 PE-conjugated	Biolegend	133906
Rat anti-mouse CD117 APC/CY7-conjugated	Biolegend	105826
RW4 ESCs	ATCC	CRL-12418