SIRPA Human, HEK

What is the molecular structure and function of SIRPA Human?

SIRPA, also known as CD172a or SHPS-1 (SHP substrate 1), is a monomeric approximately 90kDa type I transmembrane glycoprotein belonging to the SIRP/SHPS family of the immunoglobulin superfamily. It functions as an immunoglobulin-like cell surface receptor for CD47 and acts as a docking protein that induces translocation of binding partners including PTPN6 and PTPN11 from the cytosol to the plasma membrane. SIRPA is ubiquitously expressed with particularly high expression in brain tissue . The protein supports important cellular functions including adhesion of cerebellar neurons, neurite outgrowth, and glial cell attachment, potentially playing key roles in intracellular signaling during synaptogenesis and synaptic function .

How is SIRPA expression regulated during cellular differentiation?

SIRPA expression follows a specific temporal pattern during differentiation of human pluripotent stem cells. It is not detected in undifferentiated human embryonic stem cells (hESCs) or in early cardiac mesoderm (day 5 of differentiation). SIRPA expression is first observed between days 7-8 of differentiation, coinciding with the emergence of NKX2-5-positive cardiac precursor cells. The percentage of SIRPA-positive cells increases significantly over the subsequent 2-4 days and is maintained throughout further development . This temporal expression pattern makes SIRPA a valuable marker for monitoring differentiation progression, particularly in cardiac lineage development.

What detection methods are most effective for SIRPA Human?

SIRPA can be detected through multiple experimental approaches including western blot analysis, immunoprecipitation, flow cytometry, and immunofluorescence. For flow cytometry, both directly conjugated (SIRPA-PE-CY7) and biotinylated (SIRPA-bio) antibodies have proven effective in identifying SIRPA-positive populations . Notably, SIRPA-positive cells detected in differentiation cultures appear substantially larger than SIRPA-negative populations, indicating that cell size can be monitored using anti-SIRPA antibodies . For immunofluorescence analysis, anti-SIRPA antibodies reliably detect surface expression on cardiomyocytes, as verified by co-staining with cardiac Troponin I (cTNI) .

How can SIRPA be used to isolate cardiomyocytes from differentiation cultures?

SIRPA has been identified as a specific cell-surface marker for cardiomyocytes derived from both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). In a comprehensive screen against 370 known CD antibodies, SIRPA was the only marker that displayed a cardiomyocyte-specific expression pattern . Cell sorting using anti-SIRPA antibodies allows for efficient enrichment of cardiac precursors and cardiomyocytes from differentiation cultures, yielding populations containing up to 98% cardiac troponin T-positive cells . The isolated SIRPA-positive cells maintain their contracting phenotype when plated in culture and can be maintained over extended periods, making this method valuable for obtaining highly purified cardiomyocyte populations for downstream applications.

What is the relationship between SIRPA and CD47 in stem cell research?

SIRPA functions as the receptor for CD47, with this interaction playing critical roles in multiple cellular processes. In the context of stem cell research, gene expression analysis has shown that CD47 is expressed at low levels in undifferentiated ESCs and day 5 differentiation cultures, but its expression increases and becomes broadly distributed throughout the entire cell population between days 8 and 20 of differentiation . This corresponds with the emergence of SIRPA expression, suggesting coordinated regulation of this receptor-ligand pair during differentiation. The SIRPA-CD47 interaction is particularly important in xenograft applications, where species-specific polymorphisms in SIRPA determine binding affinity for human CD47 and subsequent engraftment efficiency of human cells in animal models .

How does SIRPA expression correlate with functional maturity in stem cell-derived cardiomyocytes?

SIRPA expression strongly correlates with functional cardiomyocyte identity. Immunofluorescence analysis has confirmed that SIRPA surface expression is exclusively found on cells co-expressing cardiac markers like Troponin I . When isolated via SIRPA-based cell sorting, the resulting cardiomyocyte populations exhibit contractile activity and can be maintained in long-term culture, indicating functional maturity . The correlation between SIRPA expression and cardiomyocyte functionality makes it an excellent marker not only for identifying cardiac lineage cells but also for selecting functionally mature cardiomyocytes for applications requiring contractile activity.

What methods can be used to engineer SIRPA expression in HEK cells?

While not explicitly detailed in the search results, standard molecular biology techniques can be employed to engineer SIRPA expression in HEK cells. These include transient transfection with SIRPA expression vectors, stable integration using selectable markers, or CRISPR/Cas9-mediated genome editing to either modify endogenous SIRPA or introduce exogenous variants. For functional studies, CD47 knockout HEK293T cells provide an excellent control system, as demonstrated in binding experiments with SIRPA-expressing extracellular vesicles . When designing SIRPA expression systems in HEK cells, researchers should consider including appropriate epitope tags or fluorescent reporters to facilitate detection and purification.

How can SIRPA-CD47 interactions be quantitatively assessed in HEK cell models?

Quantitative assessment of SIRPA-CD47 interactions in HEK cell models can be performed using cell binding assays. For example, fluorescently labeled SIRPA-expressing constructs (such as Cy5.5-labeled SIRPA-extracellular vesicles) can be co-incubated with CD47 knockout and wild-type HEK293T cells to measure binding specificity and affinity . Flow cytometry provides a robust method for quantifying these interactions, allowing for measurement of both percentage of binding-positive cells and binding intensity. Additionally, surface plasmon resonance or bio-layer interferometry with purified proteins can provide detailed kinetic parameters of the SIRPA-CD47 interaction.

What are the experimental considerations when using HEK cells for SIRPA-related studies?

When using HEK cells for SIRPA-related studies, several experimental considerations are important. First, the endogenous expression levels of both SIRPA and CD47 in HEK cells should be characterized, as these may influence experimental outcomes. Second, for interaction studies, appropriate controls including CD47 knockout cells are essential to confirm binding specificity . Third, researchers should consider the impact of SIRPA polymorphisms, as these can significantly affect binding affinity for CD47, particularly in cross-species studies . Finally, the subcellular localization of SIRPA should be verified using immunofluorescence or cell surface biotinylation to ensure proper membrane expression.

How can SIRPA-engineered extracellular vesicles be produced at scale?

SIRPA-engineered extracellular vesicles (SIRP-EVs) can be produced using genetically modified mesenchymal stem cells (MSCs) expressing SIRPA. A scalable production process has been developed utilizing 3D bioreactor systems that represent a significant improvement over traditional 2D culture methods . This process includes:

• Upstream production in 3D bioreactors (from small-scale Ambr250 to large-scale 50L STR systems)

• Downstream processing with:

  • Depth filter filtration for clarification

  • Tangential flow filtration for concentration

  • Chromatography for purification

  • Additional tangential flow filtration for buffer exchange and further concentration

This approach maintains consistent SIRPA expression in MSCs throughout the collection period and ensures high-purity SIRP-EVs that preserve MSC properties while achieving significant production levels .

What therapeutic applications have been explored for SIRPA-based approaches?

SIRPA-based approaches have shown promising therapeutic potential, particularly in inflammatory conditions. SIRP-EVs derived from genetically engineered MSCs have been investigated as a treatment for acute liver failure (ALF), a critical inflammatory condition characterized by rapid hepatocyte death and impaired liver regeneration . These SIRP-EVs function through a novel dual-mode action mechanism:

• They target and block CD47 (the "don't eat me" signal) on necroptotic hepatocytes, facilitating their clearance

• They simultaneously deliver MSC-derived regenerative proteins to damaged tissue

In ALF models, SIRP-EVs decreased CD47+ necroptotic cells, promoted liver regeneration, reduced liver damage markers, and enhanced survival rates . This approach may have applications in other inflammatory diseases where both clearance of damaged cells and tissue regeneration are needed.

How do SIRPA polymorphisms impact xenograft research applications?

SIRPA polymorphisms significantly impact xenograft research, particularly in human-to-mouse transplantation models. These polymorphisms determine binding affinity for human CD47, which is critical for engraftment efficiency of human cells . Researchers have developed specialized mouse models with human SIRPA knock-in (e.g., C57BL/6.Rag2nullIl2rgnull with Sirpahuman/human or BRGShuman mice) to address this issue .

Macrophages from these human SIRPA knock-in mice demonstrate:

• Significantly stronger affinity for human CD47 than those with SirpaNOD/NOD

• Absence of detectable phagocytosis against human hematopoietic stem cells

• Moderate affinity for mouse CD47, preventing blood cytopenia seen in Sirpa-/- mice

These specialized models show significantly greater engraftment and maintenance of human hematopoiesis with high levels of myeloid reconstitution compared to conventional models, making them valuable tools for studying normal and malignant human stem cells in a more authentic xenotransplantation context .

What are the critical quality control parameters for SIRPA-based cell isolation?

When using SIRPA for cell isolation, particularly cardiomyocytes, several quality control parameters are essential. Flow cytometry antibody validation should include comparison of directly conjugated and biotinylated antibodies to ensure consistent staining patterns . Western blot analysis and immunoprecipitation for SIRPA can confirm antibody specificity . Post-isolation assessment should include verification of cardiac marker expression (e.g., cardiac Troponin T or I) to confirm enrichment efficiency, which can reach up to 98% in optimized protocols . Additionally, functional assays such as contractility assessment and electrophysiological characterization provide important validation of the isolated cardiomyocyte population's physiological relevance.

How can researchers troubleshoot inconsistent SIRPA detection in differentiation experiments?

Inconsistent SIRPA detection in differentiation experiments may stem from several factors. Temporal expression patterns should be carefully considered, as SIRPA is first detected between days 7-8 of differentiation and increases over the subsequent 2-4 days . Antibody selection is crucial, with both directly conjugated (SIRPA-PE-CY7) and biotinylated (SIRPA-bio) antibodies showing reliable detection . Flow cytometry gating strategies should account for the observation that SIRPA+ cells appear substantially larger than SIRPA- populations . Differentiation protocol variations may affect the timing and efficiency of SIRPA expression, necessitating optimization for each experimental system. Finally, background control staining using appropriate isotype controls and secondary antibody-only conditions should be included in each experiment.

What are the best practices for interpreting SIRPA-CD47 functional studies?

When interpreting SIRPA-CD47 functional studies, researchers should consider several best practices. First, appropriate controls including CD47 knockout cells are essential for confirming binding specificity . Second, species-specific polymorphisms in SIRPA can significantly affect binding affinity for CD47, particularly important in cross-species studies or when using recombinant proteins from different species . Third, quantitative methods should be used to assess binding, such as flow cytometry or surface plasmon resonance, rather than relying solely on qualitative observations. Finally, functional readouts beyond binding (such as effects on phagocytosis, cell signaling, or phenotypic changes) provide more comprehensive understanding of the biological significance of SIRPA-CD47 interactions in the experimental system.