小鼠胚胎的逆转录病毒感染干细胞衍生胚体细胞造血分化的分析
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
如上所讨论的，可以使用多西环素系统产生ESC克隆诱导地表达感兴趣的基因，然而，产生这些线是耗时且劳动密集型的。另外，在本协议是为了表达从ESC准备单细胞悬液衍生胚目的基因的方法。然后，这些被感染的细胞改造成胚体由悬滴检查后续的分化。在这个例子中（ 图3），它示出了mirn23a簇的表达增强了造血发育时，在分化的第3天表达。在这一点上已发生新生胚层组织的?…
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 |
References
- Williams, R. L., et al. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature. 336, 684-687 (1988).
- Desbaillets, I., Ziegler, U., Groscurth, P., Gassmann, M. Embryoid bodies: an in vitro model of mouse embryogenesis. Experimental physiology. 85, 645-651 (2000).
- Hopfl, G., Gassmann, M., Desbaillets, I. Differentiating embryonic stem cells into embryoid bodies. Methods Mol Biol. 254, 79-98 (2004).
- Tian, X., Kaufman, D. S. Differentiation of embryonic stem cells towards hematopoietic cells: progress and pitfalls. Curr Opin Hematol. 15, 312-318 (2008).
- Thomas, K. R., Capecchi, M. R. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell. 51, 503-512 (1987).
- Mansour, S. L., Thomas, K. R., Capecchi, M. R. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature. 336, 348-352 (1988).
- Robertson, E., Bradley, A., Kuehn, M., Evans, M. Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector. Nature. 323, 445-448 (1986).
- Cherry, S. R., Biniszkiewicz, D., van Parijs, L., Baltimore, D., Jaenisch, R. Retroviral expression in embryonic stem cells and hematopoietic stem cells. Mol Cell Biol. 20, 7419-7426 (2000).
- Pfeifer, A., Ikawa, M., Dayn, Y., Verma, I. M. Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc Natl Acad Sci U S A. 99, 2140-2145 (2002).
- Smith-Arica, J. R., et al. Infection efficiency of human and mouse embryonic stem cells using adenoviral and adeno-associated viral vectors. Cloning and stem cells. 5, 51-62 (2003).
- Lakshmipathy, U., et al. Efficient transfection of embryonic and adult stem cells. Stem Cells. 22, 531-543 (2004).
- Cook, B. D., Liu, S., Evans, T. Smad1 signaling restricts hematopoietic potential after promoting hemangioblast commitment. Blood. 117, 6489-6497 (2011).
- Zafonte, B. T., et al. Smad1 expands the hemangioblast population within a limited developmental window. Blood. 109, 516-523 (2007).
- Kyba, M., Perlingeiro, R. C., Daley, G. Q. HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors. Cell. 109, 29-37 (2002).
- Galvin, K. E., Travis, E. D., Yee, D., Magnuson, T., Vivian, J. L. Nodal signaling regulates the bone morphogenic protein pluripotency pathway in mouse embryonic stem cells. J Biol Chem. 285, 19747-19756 (2010).
- Ismailoglu, I., Yeamans, G., Daley, G. Q., Perlingeiro, R. C., Kyba, M. Mesodermal patterning activity of SCL. Exp Hematol. 36, 1593-1603 (2008).
- Kyba, M., et al. Enhanced hematopoietic differentiation of embryonic stem cells conditionally expressing Stat5. Proc Natl Acad Sci U S A. 100, 11904-11910 (2003).
- Cao, M., et al. MiR-23a regulates TGF-beta-induced epithelial-mesenchymal transition by targeting E-cadherin in lung cancer cells. Int J Oncol. 41, 869-875 (2012).
- Chan, M. C., et al. Molecular basis for antagonism between PDGF and the TGFbeta family of signalling pathways by control of miR-24 expression. EMBO J. 29, 559-573 (2010).
- Huang, S., et al. Upregulation of miR-23a approximately 27a approximately 24 decreases transforming growth factor-beta-induced tumor-suppressive activities in human hepatocellular carcinoma cells. Int J Cancer. 123, (2008).
- Min, S., et al. TGF-beta-associated miR-27a inhibits dendritic cell-mediated differentiation of Th1 and Th17 cells by TAB3, p38 MAPK, MAP2K4 and MAP2K7. Genes Immun. 13, 621-631 (2012).
- Mikkola, H. K., Fujiwara, Y., Schlaeger, T. M., Traver, D., Orkin, S. H. Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo. Blood. 101, 508-516 (2003).
- Mitjavila-Garcia, M. T., et al. Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells. Development. 129, 2003-2013 (2002).
- Warr, M. R., Pietras, E. M., Passegue, E. Mechanisms controlling hematopoietic stem cell functions during normal hematopoiesis and hematological malignancies. Wiley interdisciplinary reviews. Systems biology and medicine. 3, 681-701 (2011).
- Dahl, R., Ramirez-Bergeron, D. L., Rao, S., Simon, M. C. Spi-B can functionally replace PU.1 in myeloid but not lymphoid development. Embo J. 21, 2220-2230 (2002).
- Dahl, R., et al. Regulation of macrophage and neutrophil cell fates by the PU.1:C/EBPalpha ratio and granulocyte colony-stimulating factor. Nat Immunol. 4, 1029-1036 (2003).
