Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that 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.
I. Culture of human embryonic stem cells (hESCs)
II. Preparation of hESC pellet for grafting
III. Renal capsule grafting
IV. Teratoma harvest, fixation and sectioning
I. | 80% Flex alcohol | 2 hours |
II. | 80% Flex alcohol | 1 hour |
III. | 85% Flex alcohol | 1.5 hours |
IV. | 95% Flex alcohol | 1 hour |
V. | 100% Flex alcohol | 1 hour |
VI. | 100% Flex alcohol | 1 hour |
VII. | 100% Flex alcohol | 1 hour |
VIII. | Clear-Rite 3 | 1 hour |
IX. | Clear-Rite 3 | 1 hour |
X. | Clear-Rite 3 | 1 hour |
XI. | Paraffin (TissuePrep) | 2 hours |
XII. | Paraffin (TissuePrep) | 2 hours |
V. Immunohistochemistry and analysis
I. | Xylene wash | 3-5 minutes |
II. | Xylene | 3-5 minutes |
III. | 100% alcohol | 3-5 minutes |
IV. | 100% alcohol | 3-5 minutes |
V. | 95% alcohol | 3-5 minutes |
VI. | 80% alcohol | 3-5 minutes |
VII. | Water wash (several changes) | 2 minutes |
VIII. | Gill’s Hematoxylin #3 | 2-5 minutes |
IX. | Water wash (several changes) | until water runs clear |
X. | Scott’s water (to blue nuclei) | 3 minutes or more |
XI. | Water wash (several changes) | 1-2 minutes |
XII. | Eosin Y | 1 minute |
XIII. | Water wash (several changes) | 30 seconds |
XIV. | 80% alcohol | 1 minute |
XV. | 95% alcohol | 2 minutes |
XVI. | 95% alcohol | 2 minutes |
XVII. | 100% alcohol | 3 minutes |
XVIII. | 100% alcohol (clean) | 3 minutes |
XIX. | Xylene | 2 minutes or more |
XX. | Xylene | 2 minutes or more |
VI. Representative Results
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.
Gelatin
MEF medium
KSR (Knockout Serum Replacement) medium
Collagenase IV
Avertin
Buprenorphine
Ketoprofen
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.
The authors have nothing to disclose.
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.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
2-Methyl-2-butanol | Sigma-Aldrich | 240486 | Avertin | |
2,2,2-Tribromoethanol | Sigma-Aldrich | T48402 | Avertin | |
4-0 silk sutures | Ethicon | 641G | ||
bFGF | R&D Systems | 233FB | ||
Buprenorphine | Sigma-Aldrich | B7536 | ||
Clear-Rite 3 | Fisher | 22-046-341 | ||
Collagenase IV | Sigma-Aldrich | C5138 | ||
D-MEM | Invitrogen | 11965 | ||
DPBS | Invitrogen | 14190 | ||
Dupont #5 forceps | WPI | 500233 | Kidney capsule | |
Eosin Y | Fisher | 23-245-658 | ||
Fetal Bovine Serum (Qualified) | Invitrogen | 26140-079 | ||
Flex alcohol | Fisher | 22-046344 | ||
Gill’s Hematoxylin #3 | Fisher | 22-050-204 | ||
Hemostat, straight | WPI | 501241 | General surgery | |
Iris forceps | WPI | 15914 | General surgery | |
Ketoprofen | Sigma-Aldrich | K2012 | ||
KnockOut D-MEM | Invitrogen | 10829 | ||
KnockOut Serum Replacement | Invitrogen | 10828-028 | ||
L-glutamine | Invitrogen | 25020-081 | ||
MILLICELL inserts | Millipore | PICM01250 | ||
Nonessential Amino Acids | Invitrogen | 11140-050 | ||
Operating scissors | WPI | 501754 | General surgery | |
Paraffin (TissuePrep) | Fisher | 8002-74-02 | ||
Pen/Strep | Invitrogen | 15140-122 | ||
Permount | Fisher | SP15-100 | ||
PHA | Sigma-Aldrich | L1668 | ||
Porcine gelatin | Sigma-Aldrich | G6144 | ||
ROCK inhibitor | Calbiochem | Y-27632 | ||
SCID beige mice | Charles River | 250 | ||
Scott’s Wash | Fisher | 6697-32 | Ricca Chemicals | |
Vannas spring scissors | WPI | 14003 | Kidney capsule | |
β-mercaptoethanol | Sigma-Aldrich | M7522 |