Molecular Analysis of Differentiated Cells

When hESCs or mESCs are manipulated to produce differentiated cells, tissue-specific molecular markers should be examined to confirm the lineage of generated cells. Immunocytochemistry is a convenient, yet powerful, approach to determine the differentiated cell type because the specific molecular signal and cell morphology can be simultaneously analyzed at high resolution. To further molecularly dissect the role of a signaling pathway in the cell fate determination, the luciferase assay can be used as a routine technique. This method quantifies the activity of the reporter construct driven by the tissue specific promoter (e.g., Rex-1 or Oct-3/4 for the undifferentiated hESCs or mESCs [13], Gata4 or Gata6 for early endodermal differentiation [24], Ta-1 for neuronal differentiation [25]) under the conditions in which the activity of the specific signaling pathway is altered.

3.3.1. Immunocytochemistry

We usually use fluorescence-conjugated secondary antibodies to locate the signal recognized by the primary antibody. Other methods including immu-noperoxidase can be also used to visualize the signal when the autofluorescence level of the sample is too high.

1. Aspirate the culture medium from hESCs or mESCs.

2. Fix cells in 4% paraformaldehyde for 15 min.

3. Wash cells with PBS/0.1% BSA for 5 min, three times.

4. If the antigen to be detected is cytoplasmic (e.g., intermediate filaments) or in the nucleus (e.g., transcription factors), permeabilize cells with 0.3% Triton X-100 in PBS/0.1% BSA containing 10% serum for 45 min (see Note 13). If the antigen is on the cell surface, skip this step because this treatment will severely disrupt surface antigens.

5. Add an appropriate concentration of a primary antibody dissolved in PBS/0.1% BSA/10% serum onto each well, and incubate for 1-3 h at room temperature or overnight at 4°C.

6. Wash cells with PBS/0.1% BSA for 5 min, three times.

7. Add an appropriate concentration of a fluorescence-conjugated secondary antibody dissolved in PBS/0.1% BSA/10% serum onto each well, and incubate for 45 min at room temperature (see Note 14).

8. Wash cells with PBS/0.1% BSA for 5 min, three times.

9. Observe the sample under fluorescent microscope.

3.3.2. Luciferase Reporter Assay

A dual luciferase reporter assay system is used because the experimental reporter activity (firefly luciferase) can be easily standardized by the internal control Renilla reporter activity (Renilla luciferase) in a single sample in the same tube.

1. Plate cells (mESCs; 5000 cells/cm2 or hESCs; approx 50 cells/clump, 100 clumps/cm2) on 24-well plates.

2. Transfect the firefly or Renilla reporter plasmid (mESCs; 100 ng or 10 ng per well, respectively, or hESCs; 500 ng or 20 ng per well, respectively) and specific effector constructs (100-500 ng) in triplicate by using Lipofectamine 2000 according to the manufacturer's protocol.

3. Forty-eight hours after transfection, aspirate the medium and add 100 pL of 1X passive lysis buffer to each well.

4. Gently shake the plate on an orbital shaker for 15 min.

5. Harvest the cell lysate by using a scraper and transfer it into 1.5-mL Eppendorf tubes.

6. Mix 10 pL of the lysate with 100 pL of Stop & Glo Buffer in a glass tube.

7. Measure the luciferase activity using a Lumat luminometer.

8. Standardize the reporter activity by the Renilla reporter activity.

4. Notes

1. Too many colonies in the same well would attach together during the culture period and tend to delay the differentiation process.

2. The optimal concentration of the GSK-3 inhibitor (BIO) should be predetermined for each hESCs line by testing different concentrations of BIO, generally ranging from 1 to 5 |jM. In our experience, hESCs growing in shorter doubling time require relatively lower concentration of BIO. Although BIO is exceptionally highly specific to GSK-3, other kinases, including CDKs, can be also affected by BIO treatment. It is therefore critical to find out a minimal concentration of BIO that can maintain each hESCs line in the undifferentiated state to avoid any significant effect on their viability or growth rate.

3. Some hESCs at the margin of the colony may start migrating and spreading out from the colony. Generally, hESCs in smaller colonies exhibit morphological change faster and clearer than hESCs in larger colonies, which sometimes tend to remain undifferentiated even after 5 d of differentiation period.

4. We find that after two to three passages, BIO-treated hESCs tend to reduce their growth rate and self-renewal function. We are establishing the passaging protocol that maximizes BIO-mediated self-renewal in hESCs through combining different types of chemical compound to reduce cytotoxicity of BIO. Although it is quite challenging to replace biological Wnt activity simply with the synthetic chemical compound without affecting any cell functions, we believe that developing this method is one of the first steps to provide self-renewing hESCs toward therapeutic applications.

5. For instance, in case of Lipofectamine, 5 yg of the reporter plasmid or 10 yL of Lipofectamine is dissolved in 250 yL of OptiMEM in separate Falcon 2095 tubes and incubated for 5 min. Then, mix these contents together and incubate another 20 min. Add the mixture onto mESCs in one well of a six-well plate and gently swirl the culture vessel to mix the content with the culture medium.

6. If the MESCs tend to differenciate under the drug selection conditon, MESCs should be plated on mutant mice-derived MEFs resistant to the drug used for the selection.

7. It is highly recommended to predetermine the optimal concentration of the drug to completely kill the parental nontransfected cells by 5-7 d after treatment. Timing or robustness of cell death after drug treatment is highly variable depending on types and concentrations of drugs to be used. Generally, neomycin is used at concentrations ranging of 80-200 yg/mL. Cells start dying as early as 2-4 d after treatment. In case of Puromycin, we use at 0.5-2 yg/mL. Cell death is visible as early as 1 d after treatment.

8. It is recommended to use a laminar flow hood equipped with a dissecting microscope to avoid any airborne contamination.

9. During further passaging, mESCs in several colonies sometimes start losing GFP expression even in the undifferentiated state under the drug selection. When it occurs, repeat the cloning process shown above to maintain uniformly GFP-positive mESC colonies.

10. Too many colonies in the same well may obscure the border between each colony because hESCs grow during a long period (3 wk to complete neurogenesis in hESCs).

11. After around 2 wk of the culture period, PA6 cells tend to shrink and curl up from the culture vessel. To prevent coming off of the entire hESCs colonies and feeder cells, it is important to replace the medium as gentle as possible. Pour the medium slowly while attaching the tip of the pipet to the vessel wall. Never flash the medium directly onto the cell surface.

12. Here is the example of tissue-specific antibodies. Cytokeratin (epidermis, ectoderm origin), smooth muscle actin (mesoderm origin), Tuj-1 (neuron, ectoderm origin), a-fetoprotein (liver, endoderm origin), glial fibrillary acidic protein (GFAP, astrocyte, ectoderm origin), and so on.

13. The species of the serum should be matched to the species by which the secondary antibody is raised.

14. After adding the fluorescence-conjugated secondary antibody, the culture plates should always be kept in dark to avoid photobleaching of positive signals.


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