Retroviral infektion af murine embryonale stamceller Afledt embryoide kroppens celler til analyse af Hæmatopoietisk Differentiering
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 (HPCs)4.
Gene expression in ESCs can be manipulated by several techniques that allow for the determination of a role for individual molecules in development. One of the most common techniques is to use homologous recombination to generate ESC lines, which lack a gene of interest5,6. There are also a number of techniques that have been used to overexpress genes. The first technique used to modify gene expression in ESCs was to infect them with recombinant retroviruses7,8. The gene of interest however is often silenced as the ESCs differentiate into progenitor and mature cell types. Use of lentiviruses has been successful in limiting the silencing of virally expressed genes9. Other viral vectors used for overexpression include adenovirus and adeno-associated virus10. In addition standard transfection techniques to stably introduce expression plasmids are widely used for ESC transgene expression11. One difficulty with these systems is that often expression of a specific gene has different effects depending on its temporal expression. For example, the Smad1 protein affects the development of hematopoietic cells differently during different stages of EB development12,13.
This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest. The most common system for inducibly expressing transgenes in ESCs uses the tetracycline resistance operon from E. Coli (Escherichia coli). Several different tetracycline systems have been developed. One of the more popular strategies was developed by Kyba and colleagues14. They generated an ESC line (Ainv15), which has the reverse tetracycline transactivator gene inserted into the constitutively active ROSA26 locus. A tetracycline response element (TRE), a downstream LoxP site (locus of X-over P1 from bacteriophage P1), and a promoterless neomycin cassette were introduced into the HPRT (Hypoxanthine-guanine phosphoribosyltransferase) locus on the X chromosome. Using a CRE recombination approach a gene of interest along with a eukaryotic promoter to drive the expression of the neomycin resistance gene can be inserted into the LoxP site. Correctly targeted ESCs are isolated by G418 selection. These targeted clones then must be tested for doxycycline (or tetracycline) inducible expression of the transgene. This approach has successfully generated ESC lines that inducibly express HoxB4, Stat5, SCL, and Smad714-17. However, generation of these inducible cell lines is time consuming. Described here is a method to disaggregate ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infect the cell suspensions at different days of development, and then reform the EBs by hanging drop. Downstream differentiation is later evaluated by flow cytometry. In this article an example of how miRNA expression in EB-derived cells effects hematopoietic differentiation is shown. This method is useful for investigating the role of genes after specific germ layer tissue is derived.
Protocol
Representative Results
Discussion
Som beskrevet ovenfor, kan ØSU kloner, som inducerbart udtrykker et gen af interesse genereres ved hjælp af doxycyclin systemer dog generation af disse linjer er tidskrævende og arbejdskrævende. Beskrevet i denne protokol er en metode til at udtrykke et gen af interesse i en enkelt celle suspension fremstillet fra ESC afledte EB'er. Disse inficerede celler bliver derefter reformeret i EB'er ved hængning slip for at undersøge efterfølgende differentiering. I eksemplet (figur 3)…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by a Pilot and Feasibility Grants from the Indiana University School of Medicine Center (IUSM) for Excellence in Molecular Hematology (NIDDK, 5P30DK090948). Additional funding was provided by a Biomedical Enhancement Grant from IUSM. We would like to thank Dr. Karen Cowden Dahl for commenting on the manuscript.
Materials
| Reagent | Company | Catalog Number |
| DMEM | Sigma/ Aldrich | D5796 |
| b-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 10cm | Fisher Scientific | 08-757-100D |
| Falcon Petri dish 15cm | Fisher Scientific | 08-757-148 |
| Falcon Tube 5mls | Fisher Scientific | 14-959-11A |
| Falcon Tube 5mls 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 |
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