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Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers. This makes them a potentially valuable tool for the treatment of many different diseases. With the advent of induced pluripotent stem cells (iPSCs) and continuing research with human embryonic stem cells (hESCs) there is a need for assays that can demonstrate that a particular cell line is pluripotent. Germline transmission has been the gold standard for demonstrating the pluripotence of mouse embryonic stem cell (mESC) lines1,2,3. Using this assay, researchers can show that a mESC line can make all cell types in the embryo including germ cells4. With the generation of human ESC lines5,6, the appropriate assay to prove pluripotence of these cells was unclear since human ESCs cannot be tested for germline transmission. As a surrogate, the teratoma assay is currently used to demonstrate the pluripotency of human pluripotent stem cells (hPSCs)7,8,9. Though this assay has recently come under scrutiny and new technologies are being actively explored, the teratoma assay is the current gold standard7. In this assay, the cells in question are injected into an immune compromised mouse. If the cells are pluripotent, a teratoma will eventually develop and sections of the tumor will show tissues from all 3 germ layers10. In the teratoma assay, hPSCs can be injected into different areas of the mouse. The most common injection sites include the testis capsule, the kidney capsule, the liver; or into the leg either subcutaneously or intramuscularly11. Here we describe a robust protocol for the generation of teratomas from hPSCs using the testis capsule as the site for tumor growth.
Note: All animal procedures must be approved by IACUC or equivalent.
All surgical equipment must be sterilized prior to surgery. Sterile gloves, drapes and gauze must be used.
When this protocol is done as described and the injected cell line is pluripotent, a palpable, visually obvious, tumor should form within 12 weeks at the most. For established hPSC lines like WA09, we typically see tumors within 6 weeks. For iPSC lines it is not unusual to see tumors within 8-10 weeks. It is very important to inject mice with a line that is known to be pluripotent, as a positive control, in order to be sure that the procedure was performed correctly. Tumors usually look very heterogeneous and have many attached cysts (Figure 1). Analysis of the tumor samples by a pathologist should show differentiated tissues from all three germ layers (Figure 2).
Figure 1. Typical hPSC derived teratoma in the testis capsule.
Following the described protocol, one million WA09 cells were injected into the testis capsule of an immune compromised mouse. Six weeks later a teratoma was observed. A) Teratoma pulled from the mouse. B) Close up picture of the teratoma. Note the heterogeneity and cyst structures.
Figure 2. Hematoxylin and Eosin stained sections from the teratoma show tissues from each germ layer. Following fixation, the teratoma was sectioned and stained with Hematoxylin and Eosin. Analysis by a pathologist revealed the presence of cells from each of the 3 germ layers.
The method presented here provides a highly reliable, straightforward means of generating teratomas from hPSCs in the testis capsule. There are several critical parameters in this technique. In particular, it is important to inject hPSC cell lines that are known to be pluripotent as a control. Other important parameters include the time interval between injection and tumor observation. For cell lines like WA09, teratomas should be observed in 6-8 weeks. For new iPSC lines we find that often 10 weeks are required. Another concern is the number of cells injected. We inject one million cells but the assay can easily be done with fewer cells. In addition, the injection medium is important. We find that we get the best results when the cells are injected in a 1:1 mixture of matrigel and DMEM/F-12, as opposed to PBS or DMEM/F-12.
No conflicts of interest declared.
This work was funded by CIRM grants #TR-1250, RT1-01108, and CL1-00502.