CD117 Antibody: Key Factor in Hematopoietic Stem Cell Studies

CD117 antibody is widely used in biomedical research, particularly in the study of hematopoietic stem cells (HSCs). Its significance lies in its ability to bind specifically to CD117, a receptor that plays a key role in cell signaling and development. Researchers use this antibody to identify and isolate HSCs, which are crucial for understanding blood formation and potential therapeutic applications.

Scientists have developed various methods to detect CD117 expression in different cell types, refining knowledge of stem cell biology and disease mechanisms.

The CD117 Receptor

CD117, also known as c-Kit, is a transmembrane receptor tyrosine kinase that plays a fundamental role in cellular signaling, particularly in hematopoiesis. Encoded by the KIT gene on chromosome 4q12, this receptor regulates stem cell maintenance, proliferation, and differentiation. It is activated by stem cell factor (SCF), which induces receptor dimerization and autophosphorylation, triggering downstream signaling cascades such as PI3K/AKT, RAS/MAPK, and JAK/STAT. These pathways influence cell survival, migration, and lineage commitment, making CD117 essential in HSC development.

CD117 expression is tightly regulated during hematopoiesis, with high levels in early progenitor cells that diminish as differentiation progresses. This dynamic expression allows researchers to use CD117 as a biomarker for isolating HSCs from bone marrow, umbilical cord blood, and peripheral blood. Beyond hematopoietic cells, CD117 is found in melanocytes, germ cells, and interstitial cells of Cajal, contributing to tissue homeostasis and cellular communication. Mutations in the KIT gene can lead to aberrant signaling implicated in disorders such as mastocytosis, gastrointestinal stromal tumors (GISTs), and certain leukemias.

CD117’s structure underscores its functional significance. The extracellular domain consists of five immunoglobulin-like loops responsible for SCF binding, while the intracellular region contains a split kinase domain essential for enzymatic activity. Mutations in these domains can result in either loss- or gain-of-function alterations, with the latter often linked to oncogenic transformation. KIT mutations in exon 11 are common in GISTs, leading to constitutive receptor activation and uncontrolled proliferation. Targeted therapies, such as tyrosine kinase inhibitors (TKIs) like imatinib, have been developed to counteract these aberrant signals, demonstrating CD117’s therapeutic relevance in oncology.

Production And Structure Of CD117 Antibody

CD117 antibodies are developed using sophisticated techniques to ensure specificity and high affinity for the c-Kit receptor. Monoclonal antibodies, the most commonly used format, are generated through hybridoma technology or recombinant protein engineering. In hybridoma production, mice are immunized with recombinant CD117 protein or peptide fragments, stimulating an immune response that generates B cells producing target-specific antibodies. These B cells are fused with myeloma cells to create hybridomas, which are screened for optimal binding properties. Recombinant antibody production uses phage display or single-cell sequencing to identify and express antibody genes in mammalian or bacterial systems, allowing precise engineering of binding characteristics.

Once produced, CD117 antibodies undergo validation to confirm specificity and functionality. Enzyme-linked immunosorbent assays (ELISA) quantify binding affinity, surface plasmon resonance (SPR) assesses kinetic interactions, and epitope mapping ensures recognition of the correct CD117 domain. Some variants recognize extracellular epitopes, making them suitable for flow cytometry and immunohistochemistry, while others target intracellular regions for applications such as western blotting. The choice of epitope is crucial in therapeutic antibody development, as blocking antibodies must interfere with ligand interaction or receptor activation to be effective.

Most CD117 antibodies are of the IgG subclass, providing stability and compatibility with secondary detection reagents. Humanized and fully human antibodies have been developed to reduce immunogenicity in clinical applications, particularly for treating CD117-driven malignancies. Engineering efforts have also produced bispecific antibodies targeting CD117 and other surface markers, improving selectivity in stem cell isolation or cancer treatment. Modifications such as Fc region alterations enhance functions like antibody-dependent cellular cytotoxicity (ADCC), broadening their potential beyond research.

Role In Hematopoietic Stem Cell Identification

CD117 antibody is essential in identifying hematopoietic stem cells (HSCs) due to its ability to selectively bind c-Kit, a receptor expressed on early progenitor cells. Unlike lineage-committed cells that gradually lose CD117 expression, true HSCs retain this marker alongside others such as CD34, particularly in human samples. This makes CD117 a cornerstone in isolating and characterizing stem cells from bone marrow, peripheral blood, and umbilical cord blood, aiding both research and clinical applications.

CD117 is also used to selectively enrich stem cell populations for transplantation and regenerative medicine. Fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) leverage CD117 antibody binding to separate HSCs from heterogeneous cell mixtures, improving stem cell purity. This is particularly valuable in bone marrow transplantation, where precise isolation of functional HSCs enhances engraftment efficiency and reduces transplant failure. CD117-positive HSCs exhibit superior self-renewal and multilineage differentiation potential compared to CD117-negative counterparts, underscoring the biological significance of this marker.

Beyond transplantation, CD117 expression provides insights into hematopoietic disorders originating from stem cell dysfunction. Myelodysplastic syndromes (MDS) and certain leukemias often involve aberrant CD117 signaling, leading to impaired differentiation and unchecked proliferation. Tracking CD117 levels in patient samples helps clinicians assess disease progression and therapeutic response. Experimental models using CD117 antibody-based isolation have revealed critical aspects of stem cell aging, niche interactions, and hematopoietic reconstitution after injury or chemotherapy, contributing to targeted therapies aimed at restoring normal hematopoiesis.

Expression In Mast Cells And Other Cell Types

CD117 is highly expressed on mast cells, where it regulates development, survival, and function. Mast cells originate from hematopoietic progenitors and rely on CD117 signaling for proper maturation and tissue distribution. Interaction with stem cell factor (SCF) promotes proliferation and prevents apoptosis. Dysregulation of CD117 in mast cells is implicated in disorders such as systemic mastocytosis, where KIT mutations lead to uncontrolled cell growth and accumulation in various tissues.

Beyond mast cells, CD117 is found in melanocytes, germ cells, and interstitial cells of Cajal. In melanocytes, it regulates pigmentation by controlling cell migration and survival. Germ cells, particularly primordial germ cells, express CD117 during early embryogenesis, facilitating migration to the developing gonads. Interstitial cells of Cajal (ICCs), which function as pacemaker cells in the gastrointestinal tract, rely on CD117 signaling for differentiation and motility regulation. Mutations affecting CD117 in these cells are associated with gastrointestinal stromal tumors (GISTs), highlighting its role in maintaining normal cellular function across multiple tissues.

Laboratory Detection Methods

Detecting CD117 expression requires specialized techniques that provide qualitative and quantitative insights. These methods assess CD117’s presence, distribution, and functional status in hematopoietic stem cells, mast cells, and tumor samples. Using antibodies that specifically bind the receptor, researchers analyze CD117 expression patterns in normal and diseased tissues, aiding in diagnosis and therapeutic applications. The three most widely used detection techniques are flow cytometry, immunohistochemistry, and western blotting.

Flow Cytometry

Flow cytometry analyzes CD117 expression at the single-cell level, making it useful for identifying hematopoietic stem cells and progenitor populations. Fluorescently labeled CD117 antibodies bind to the receptor on live or fixed cells, enabling rapid, high-throughput analysis. Combining CD117 staining with other surface markers such as CD34 and lineage-specific antigens allows researchers to distinguish HSCs from differentiated cells in bone marrow or peripheral blood samples.

The sensitivity of flow cytometry detects subtle variations in CD117 expression, which is critical for assessing stem cell heterogeneity and disease states. In leukemia research, abnormal CD117 expression can indicate malignant progenitor cells, aiding classification and prognosis. This technique also evaluates targeted therapies that modulate CD117 signaling, monitoring treatment response at the cellular level. Advances in spectral and mass cytometry allow researchers to analyze CD117 alongside numerous other markers, providing deeper insights into stem cell biology and tumor microenvironments.

Immunohistochemistry

Immunohistochemistry (IHC) visualizes CD117 expression in tissue sections, providing spatial context not possible with flow cytometry. Enzyme-linked or fluorescently conjugated antibodies detect CD117 in formalin-fixed, paraffin-embedded samples, making IHC valuable in research and clinical pathology. Examining CD117 distribution in bone marrow biopsies, gastrointestinal tumors, and skin lesions helps assess normal and abnormal localization.

In gastrointestinal stromal tumors (GISTs), IHC staining for CD117 is a standard diagnostic criterion, as most of these tumors exhibit strong c-Kit expression. Staining intensity and pattern—whether membranous, cytoplasmic, or punctate—provide clues about tumor subtype and responsiveness to tyrosine kinase inhibitors like imatinib. IHC is also used in mast cell disorders, where diffuse CD117 expression differentiates systemic mastocytosis from reactive mast cell hyperplasia.

Western Blot

Western blotting detects and quantifies CD117 protein in cell lysates or tissue extracts. This method separates proteins by gel electrophoresis, transfers them onto a membrane, and probes with CD117-specific antibodies. Unlike flow cytometry or IHC, western blotting confirms molecular weight, distinguishing full-length CD117 from truncated or modified variants.

This approach is useful for studying CD117 signaling dynamics, assessing receptor phosphorylation after stem cell factor (SCF) stimulation. Phospho-specific antibodies determine whether CD117 is actively engaged in downstream signaling pathways. Western blotting also evaluates tyrosine kinase inhibitors’ effects on CD117 activity, providing mechanistic insights into therapeutic efficacy.