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Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.
The written protocol below is based on published parameters reported by the authors, with some modifications. The protocol is performed with approval by the UCSF Institutional Animal Care and Use (IACUC) and Stem Cell Research Oversight (SCRO) Committees.
Figure 1. Plating of irradiated CF1 mouse embryonic fibroblasts as a feeder layer for culturing undifferentiated hESCs. MEFs are plated at ~50% confluence, as shown here, or 4 x 105 cells per 3.5 cm diameter well in a 6-well culture dish. Bar, 100 μm.
Figure 2. Example of subconfluent culture of undifferentiated hESCs on a MEF feeder layer. hESCs are grown on MEF feeder layers until ~70% confluent, as shown here, then passaged as described. Bar, 100 μm.
Figure 3. Example of explanted teratomas. Following explantation, the teratoma, kidney, and any accessory tissue are fixed as a block in formalin, then the kidney and accessory tissue are carefully trimmed away before embedding in paraffin.
Figure 4. Teratomas derived from undifferentiated hESCs contain tissues representative of all three germ layers in vivo. A teratoma, such as one depicted in Figure 3, was sectioned and stained with hematoxylin and eosin to identify embryonic tissues. hESCs give rise to tissues derived from all three embryonic germ layers (ectoderm (A,B), mesoderm (C), endoderm (D). (A) Nascent neural tube structure. (B) Primitive squamous epithelium. (C) Cartilage surrounded by capsule of condensed mesenchyme. (D) Glandular intestinal structure. Bar, 100 μm. Images reprinted with permission of author.
Figure 5. Teratoma analysis can be used to map the fate of tagged, undifferentiated hESCs. As previously published (9), a mixture of specifically tagged hESCs with untagged hESCs can be used to map the fate of the tagged cells in a teratoma formation assay. As shown here, undifferentiated hESCs expressing the surface marker, CD133, and tagged with enhanced green fluorescent protein (eGFP), were mixed with untagged, undifferentiated hESCs. These were used to form teratomas by renal capsule grafting. Immunohistochemical analysis of hematoxylin and eosin-stained teratoma sections with anti-eGFP antibody demonstrates the neuroectodermal fate of the CD133+ hESCs. Teratomas were processed as in Figure 4 and counterstained with anti-eGFP antibody (brown) to localize derivatives of CD133+GFP+ cells within tissues. CD133+-derived cells (arrows) were observed in tissues arising from embryonic ectoderm, specifically neural epithelium (left) and nascent neural tube-like structures (right). Bar, 100 μm. Images reprinted with permission of author.
KSR (Knockout Serum Replacement) medium
We have adapted a highly efficient teratoma formation assay for the purpose of mapping the fate of differentiating hESCs in an in vivo environment. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. The fate of the originally selected hESCs can then be determined by immunohistochemistry (Figure 5), as previously reported (9). This method provides a valuable tool for identifying and generating tissue-specific reagents for cell-based therapy.
No conflicts of interest declared.
This work was supported by a Comprehensive Research Grant from the California Institute for Regenerative Medicine (RC1-00104), a Public Health Service Grant (HL085377) from NHLBI, and a gift from the Pollin Foundation to H.S.B.