SESA5 Antibody

What is SSEA-5 antibody and what specific antigen does it recognize?

SSEA-5 is a monoclonal antibody (clone 8e11) raised against human embryonic stem cells (hESCs) that binds a previously unidentified antigen highly and specifically expressed on human pluripotent stem cells. This antigen was identified as the H type-1 glycan, a surface carbohydrate structure . Unlike antibodies that target protein antigens, SSEA-5 recognizes a glycan structure on the cell surface, which explains why immunoprecipitation followed by mass spectrometry was unsuccessful in identifying a single peptide antigen . When researchers performed SDS-page gel electrophoresis of immunoprecipitated hESC membranes, multiple bands were visualized at approximately 127 kDa and greater than 190 kDa, indicating that the antigen is not a single protein .

How does SSEA-5 expression change during stem cell differentiation?

SSEA-5 shows remarkable specificity for undifferentiated cells. Flow cytometry analysis demonstrates that differentiation of hESCs results in a 2-3 orders of magnitude reduction in SSEA-5 binding signal, which is substantially greater than the reduction observed with established markers like TRA-1-81, SSEA-3, and SSEA-4 . This dramatic decrease makes SSEA-5 an excellent marker for monitoring pluripotency status. When hESCs were differentiated for 3 days using retinoic acid (RA) or bone morphogenetic protein 4 (BMP4), the population of cells co-expressing high levels of multiple pluripotency surface markers including SSEA-5 decreased from 52% to 6% .

Where is SSEA-5 expressed in human tissues outside of pluripotent stem cells?

Immunohistochemistry studies of a panel of 12 human tissues from 7-month-old fetuses revealed that SSEA-5 is not significantly expressed in any of the tested tissues . Additionally, SSEA-5 did not bind to in vitro differentiated hESC-derived hematopoietic CD34+CD43+ precursors, but instead labeled a distinct undifferentiated SSEA-5+CD34-CD43- population . This high specificity for pluripotent cells makes SSEA-5 particularly valuable for research and clinical applications focused on identifying residual undifferentiated cells.

How can SSEA-5 antibody be used to remove teratoma-forming cells from differentiated cell populations?

For more complete removal of teratoma-forming cells, researchers should combine SSEA-5 with additional pluripotency surface markers (PSMs). The table below summarizes experimental results using different marker combinations:

*Small growths without evidence of three germ layers

What is the optimal methodology for using SSEA-5 in immunohistochemistry?

For immunohistochemistry staining using SSEA-5 antibody, researchers should follow standard protocols for glycan epitopes. The antibody successfully labels epithelial cells expressing SSEA-4 and epithelial specific antigen (ESA) in teratomas, comprising approximately 2% of total cells as confirmed by flow cytometry . When staining teratomas, SSEA-5+ structures exhibit morphology reminiscent of primordial hPSCs, suggesting they may represent teratoma stem cells.

For optimal results, use freshly fixed tissues and avoid harsh fixation methods that might disrupt glycan structures. Standard paraformaldehyde fixation followed by paraffin embedding is suitable, but antigen retrieval steps should be carefully optimized as glycan epitopes can be sensitive to processing conditions.

How should SSEA-5 be used in flow cytometry applications?

For flow cytometry applications, SSEA-5 antibody performs optimally when used at saturating concentrations determined by titration. The antibody works well in multicolor flow cytometry panels alongside other PSMs. When performing multicolor analysis, a single population co-expressing high levels of PSMs (such as CD9, CD50, CD90, and SSEA-5) can be clearly identified and tracked during differentiation .

For accurate sorting of SSEA-5 positive and negative populations, researchers should:

• Include appropriate isotype controls

• Use viability dyes to exclude dead cells

• Set gates based on clear separation between positive and negative populations

• Consider including additional markers (CD9, CD90, CD50, CD200) for more complete removal of undifferentiated cells

How do SSEA-5 expression patterns compare across different human pluripotent stem cell lines?

Analysis has shown that SSEA-5 expression patterns are consistent across multiple hPSC lines. The hESC lines H7 and HES2, as well as hiPSC lines iPS(IMR-90), IPS(BL), and IPS(SH) all exhibited similar PSM patterns including SSEA-5 expression . This consistency across different cell lines makes SSEA-5 a reliable marker for pluripotency regardless of the specific hPSC line being used.

For researchers working with new or uncommon hPSC lines, it is advisable to validate SSEA-5 expression patterns through flow cytometry and compare them with established pluripotency markers such as SSEA-3, SSEA-4, and TRA-1-81 before designing depletion strategies.

What is the relationship between SSEA-5 expression and teratoma formation potential?

Experimental evidence demonstrates a strong correlation between SSEA-5 expression and teratoma formation potential. When sorted for SSEA-5 expression, SSEA-5+ cells isolated from heterogeneous cultures consistently formed larger teratomas compared to SSEA-5- cells . Specifically, teratomas derived from SSEA-5-high cells were substantially larger at 4 and 6 weeks compared to those from SSEA-5-low cells (P=0.036 and P=0.049, respectively) .

What are the limitations of using SSEA-5 alone for eliminating teratoma risk?

While SSEA-5 is highly specific for undifferentiated cells, it has several limitations when used alone:

• Complete removal of teratoma-forming cells requires combination with additional PSMs. Depletion with SSEA-5 alone dramatically reduced teratoma-initiation potential but did not completely eliminate it .

• Some SSEA-5-low cells may still retain pluripotency and teratoma-forming potential, particularly in early stages of differentiation. These cells may not have concluded their exit from pluripotency and require detection with additional PSMs .

• The immunodepletion approach may require optimization of the PSM panel to avoid removal of desired progenies in case they express SSEA-5 or other PSMs used in the depletion strategy .

• Teratomas formed from SSEA-5-low populations, though smaller, still exhibited tissues from all three germ layers, indicating residual pluripotent cells .

How can researchers quantitatively analyze SSEA-5 expression during differentiation protocols?

Quantitative analysis of SSEA-5 expression during differentiation can be performed using flow cytometry with the following methodology:

• Collect cells at different time points during differentiation (e.g., day 0, 3, 7, 10)

• Stain with SSEA-5 antibody and additional pluripotency markers

• Analyze using flow cytometry software (such as FlowJo)

• Quantify the percentage of SSEA-5+ cells and the median fluorescence intensity (MFI)

• Plot the dynamic changes in expression over time

For greater analytical depth, researchers can perform quantitative real-time PCR on SSEA-5-sorted populations to correlate surface marker expression with pluripotency gene expression (POU5F1/OCT3/4, NANOG, SOX2) . This allows for confirmation that SSEA-5 binding correlates with transcriptional pluripotency status.

What controls should be included when validating SSEA-5 antibody specificity?

To validate SSEA-5 antibody specificity, researchers should include the following controls:

• Positive controls: Undifferentiated hESCs or hiPSCs known to express SSEA-5

• Negative controls: Fully differentiated cells (e.g., after 2 weeks of RA treatment)

• Isotype controls: Appropriate isotype matched antibody controls

• Blocking controls: Pre-incubation with purified H type-1 glycan to block specific binding

• Cross-reactivity tests: Testing on multiple cell types including adult stem cells and differentiated tissues

• Comparative analysis: Side-by-side comparison with established pluripotency markers (SSEA-3, SSEA-4, TRA-1-81)

How should researchers design experiments to demonstrate the functional utility of SSEA-5-based cell separation?

A comprehensive experimental design to demonstrate the functional utility of SSEA-5-based cell separation should include:

• Spiking experiments:

  • Spike undifferentiated hPSCs into differentiated cell populations at defined ratios

  • Perform SSEA-5-based depletion and control sorting

  • Transplant into immunodeficient mice

  • Monitor teratoma formation using in vivo imaging (e.g., luciferase)

• Heterogeneous culture experiments:

  • Create partially differentiated cultures (e.g., 3-day RA treatment)

  • Sort based on SSEA-5 expression alone and in combination with other PSMs

  • Transplant sorted populations

  • Compare teratoma formation rates and characteristics

• In vitro functional characterization:

  • Assess pluripotency gene expression in sorted populations

  • Perform in vitro differentiation assays on sorted populations

  • Evaluate spontaneous differentiation potential

How does SSEA-5 compare to other established pluripotency markers?

SSEA-5 offers several advantages over traditional pluripotency markers:

• SSEA-5 shows a 2-3 orders of magnitude reduction in binding signal upon differentiation, which is substantially greater than the reduction seen with established markers like TRA-1-81, SSEA-3, and SSEA-4 .

• Unlike TRA-1-81/SSEA-4 combinations, which failed to effectively separate teratoma-forming cells (as shown in the table in section 2.1, where TRA-1-81/SSEA-4 low populations still formed teratomas in 6/6 cases), SSEA-5 in combination with additional markers (CD9/CD90 or CD30/CD200) successfully eliminated teratoma formation in most cases .

• SSEA-5 has higher specificity for pluripotent cells, as evidenced by immunohistochemistry studies showing minimal expression in fetal tissues .

What combinatorial marker strategies involving SSEA-5 are most effective for complete removal of undifferentiated cells?

Based on experimental evidence, the most effective combinatorial strategies are:

• SSEA-5/CD9/CD90: When cells from heterogeneously differentiated cultures were depleted of cells expressing high levels of these three markers, only 2 out of 6 replicates formed small tumors, while 4 out of 6 formed no tumors at all . Importantly, the small growths that did form lacked evidence of three germ layers, indicating they were not true teratomas.

• SSEA-5/CD30/CD200: Similarly effective, with only 1 out of 4 replicates forming small growths, while 3 out of 4 showed no tumor formation .

These combinatorial approaches are significantly more effective than using SSEA-5 alone or traditional marker combinations like TRA-1-81/SSEA-4, which failed to effectively remove teratoma-forming cells.

How do antibody-based methods like SSEA-5 compare to genetic methods for pluripotency assessment?

Antibody-based methods using SSEA-5 offer several advantages over genetic methods:

• Non-destructive analysis: Cells remain viable and can be used for subsequent experiments or therapeutic applications.

• Immediate assessment: Surface marker analysis provides real-time information about pluripotency status without the delay required for genetic analysis.

• Functional separation: Beyond assessment, antibody methods allow for physical separation of pluripotent and differentiated populations.

• No genetic manipulation: Avoids potential risks associated with genetic modifications required for reporter systems.

What are common challenges in SSEA-5 antibody use and how can they be addressed?

Common challenges when working with SSEA-5 antibody include:

• Epitope sensitivity: As SSEA-5 recognizes a glycan structure (H type-1), certain fixation or permeabilization methods may alter epitope accessibility. Solution: Test multiple fixation protocols and minimize exposure to harsh conditions.

• Variable expression intensity: The dynamic range of SSEA-5 expression during differentiation may make gating difficult. Solution: Include undifferentiated and differentiated controls in each experiment to establish appropriate gates.

• Background in certain cell types: Some differentiated cells may express low levels of H type-1 glycan. Solution: Use additional markers in combination with SSEA-5 for more specific identification of pluripotent cells.

• Optimization for different hPSC lines: Different cell lines may show varying levels of SSEA-5 expression. Solution: Titrate antibody concentrations for each cell line and establish baseline expression patterns.

How should researchers interpret discrepancies between SSEA-5 expression and other pluripotency indicators?

When discrepancies arise between SSEA-5 expression and other pluripotency indicators:

• Consider the kinetics of marker loss during differentiation. Surface glycans like SSEA-5 may be lost at different rates than transcription factors or other surface proteins.

• Evaluate the functional pluripotency of sorted populations using in vitro differentiation assays and in vivo teratoma formation assays.

• Assess the homogeneity of your cell population, as heterogeneous differentiation can lead to varying marker expression patterns.

• Check for technical issues with antibody staining, including proper titration, fresh antibody preparation, and appropriate controls.

• Consider cell-cycle effects, as some pluripotency markers show cell-cycle dependent expression patterns.

What are promising areas for further development of SSEA-5-based technologies?

Several promising research directions for SSEA-5-based technologies include:

• Development of clinical-grade SSEA-5 antibodies for use in purification of therapeutic cell products derived from hPSCs.

• Creation of automated systems for rapid and standardized SSEA-5-based depletion of undifferentiated cells from clinical products.

• Investigation of the functional role of H type-1 glycan (the SSEA-5 antigen) in pluripotency maintenance and differentiation.

• Development of non-antibody based detection methods for the SSEA-5 antigen, such as aptamers or small molecules, which may offer advantages for certain applications.

• Integration of SSEA-5 detection into real-time monitoring systems for manufacturing processes involving hPSC differentiation.

How might SSEA-5 contribute to improved safety of stem cell-based therapies?

SSEA-5 has significant potential to improve the safety of stem cell-based therapies through:

• Reduced teratoma risk: By enabling more complete removal of undifferentiated cells, SSEA-5-based purification can minimize the risk of teratoma formation in recipients of stem cell-derived therapies .

• Standardized safety assessment: SSEA-5 can serve as a standardized marker for quality control of cell therapy products, allowing for consistent assessment of residual undifferentiated cells.

• Combinatorial safety strategies: When used in combination with other PSMs, SSEA-5-based depletion provides a more robust safety measure than single marker approaches .

• Process validation: SSEA-5 expression analysis throughout differentiation protocols can help validate and optimize manufacturing processes for consistent removal of undifferentiated cells.

As stated by the researchers who developed SSEA-5, "It is our hope that SSEA-5 and the additional PSMs would be immediately applied to advance hPSC research and ensure the safety of patients undergoing clinical trials utilizing hPSC-derived therapeutics" .