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CD34

CD34 is a transmembrane phosphoglycoprotein belonging to the sialomucin family, primarily recognized as a cell surface marker for hematopoietic stem and progenitor cells (HSPCs), which are essential for blood cell formation. First identified in on human hematopoietic progenitors, CD34 has an apparent molecular weight of 90–170 kDa due to extensive O-glycosylation and sialylation of its extracellular domain, which includes a heavily glycosylated region, a cysteine-bonded globular domain, a juxtamembrane stalk, a single transmembrane , and a short cytoplasmic with phosphorylation sites. Beyond its role as a marker, CD34 functions in , signaling, and migration, facilitating interactions between HSPCs and the microenvironment to support hematopoiesis and prevent differentiation. It is expressed not only on HSPCs but also on diverse progenitors, including endothelial cells, mesenchymal stromal cells, muscle satellite cells, and epithelial precursors, highlighting its broader involvement in tissue regeneration and vascular development. Clinically, CD34 expression is leveraged to isolate and enrich stem cells for bone marrow and peripheral blood transplantation, a standard practice in treating hematologic malignancies and disorders. Additionally, CD34-positive cells are explored in for applications such as cardiac repair post-myocardial , , and emerging trials for vision restoration in (as of 2024), due to their pro-angiogenic properties. In , CD34 marks cancer stem cells in leukemias and solid tumors like , contributing to tumor initiation, metastasis, and resistance to therapy.

Discovery and Structure

Discovery

The CD34 was first identified in 1984 by Civin and colleagues, who developed the My10 against the KG-1a human cell line; this antibody recognized a surface expressed on approximately 1% to 4% of normal cells and enriched for hematopoietic colony-forming progenitors . The 's specificity for immature hematopoietic cells was confirmed through binding studies showing reactivity with early myeloid and lymphoid precursors but not mature leukocytes. Following its initial description, the antigen was formally designated CD34 during the early Human Leukocyte Differentiation Antigen (HLDA) workshops in the mid-, standardizing for leukocyte surface molecules identified by monoclonal antibodies. In the late and , emerged as a key method to isolate CD34+ cells, revealing their primitive nature; transplantation assays in non-human primates, such as lethally irradiated baboons, demonstrated that purified CD34+ cells could reconstitute multilineage hematopoiesis long-term. Similar results in models, including SCID-hu assays, further validated CD34 as a marker for cells capable of sustained engraftment and differentiation into multiple blood lineages. Key milestones in the 1990s included the mapping of the CD34 gene to chromosome 1q32.1 via in situ hybridization. In 1992, Simmons et al. purified and cloned the CD34 cDNA from a human genomic library, establishing its sequence and confirming its expression as a heavily glycosylated transmembrane sialomucin.

Molecular Structure

CD34 is a type I transmembrane sialomucin glycoprotein encoded by the CD34 gene, consisting of 385 amino acids with a predicted unglycosylated molecular weight of approximately 40 kDa; however, due to extensive post-translational glycosylation, the mature protein exhibits an apparent molecular weight of 110-120 kDa on SDS-PAGE. The protein's domain organization includes an extracellular N-terminal mucin-like domain spanning residues 32-162, which is rich in serine and threonine residues serving as sites for O-linked glycosylation, followed by a cysteine-rich hydrophilic region (residues 163-237) containing three potential disulfide-bonded loops. This extracellular portion is anchored by a single transmembrane domain (residues 282-302) and terminates in a cytoplasmic tail of 83 amino acids (residues 303-385) that lacks canonical signaling motifs such as ITAM or ITIM sequences. The CD34 gene is located on the long arm of human at position q32.1, spanning approximately 26 kb and comprising 8 exons that encode the full-length protein. The promoter region of the CD34 gene contains binding sites for key hematopoietic transcription factors, including GATA-2, which contributes to its regulated expression in and cells. Post-translational modifications play a critical role in CD34's structure and function, with the protein undergoing extensive O-linked and N-linked , as well as sialylation primarily on the mucin-like domain, resulting in a highly negatively charged surface. Additionally, sulfation of residues and moieties further enhances this negative charge, which is essential for modulating interactions with ligands such as . These modifications collectively contribute to the protein's sialomucin classification and its role in processes.

Expression Patterns

Hematopoietic Expression

CD34 is highly expressed on long-term hematopoietic stem cells (LT-HSCs), defined phenotypically as CD34+ CD38-, which represent the most primitive subset capable of multilineage reconstitution and self-renewal. In contrast, expression levels decrease on lineage-committed progenitors, such as multipotent progenitors (MPPs) marked as Lin- CD34+ CD38- CD90- CD45RA-, which exhibit reduced self-renewal potential compared to LT-HSCs. This hierarchical pattern underscores CD34's utility as a marker for enriching primitive hematopoietic cells during isolation procedures. During embryonic development, CD34 expression peaks in the aorta-gonad-mesonephros (AGM) region around E10-E11 in mice, coinciding with the emergence of definitive HSCs from hemogenic endothelium along the ventral wall of the dorsal aorta. This transient upregulation facilitates the initial specification and intra-aortic clustering of hematopoietic progenitors before their migration to fetal liver and subsequent seeding into bone marrow niches postnatally. In adult bone marrow, CD34 persists at lower but stable levels within perivascular and endosteal niches, maintaining HSC quiescence and long-term repopulation capacity. Quantitatively, CD34+ cells constitute approximately 1-1.5% of bone marrow mononuclear cells, providing a practical for enrichment in clinical and research settings. These cells are routinely isolated using multicolor panels incorporating CD34 alongside , , and CD45RA to delineate LT-HSCs (CD34+ - CD90+ CD45RA-) from downstream progenitors, enabling high-purity sorting for transplantation or functional assays. Developmentally, CD34 expression is upregulated in hematopoietic stem and progenitor cells (HSPCs) from fetal liver and fetal relative to adult , with fetal sources exhibiting higher frequencies of CD34+ cells—often 5-6% in early fetal blood compared to 1-1.5% in adults—reflecting greater proliferative potential and primitive composition. This elevated expression supports robust hematopoiesis during gestation, transitioning to more restricted patterns in postnatal marrow.

Non-Hematopoietic Expression

CD34 is prominently expressed on endothelial progenitor cells (EPCs) and mature endothelial cells, particularly in small vessels. This expression is observed in the , where CD34 mRNA is detectable in placental tissue and associated endothelial cells. In hair follicles, CD34 marks stem cells in the bulge region, contributing to skin homeostasis. Beyond vascular , CD34 is found in mesenchymal stem cells (MSCs), where it serves as a marker, especially in association with vasculature, challenging the traditional view of CD34 negativity in these cells. It is also expressed by fibroblasts, such as spindle-shaped stromal cells in , and neural progenitors, including those differentiating into microglia-like cells in the . Expression levels are low in kidney glomerular endothelium and podocytes, as well as in broader tissues outside specific stromal or follicular sites. In pathological conditions, CD34 expression is upregulated in inflamed tissues, where it facilitates immune and contributes to responses, such as in and bowel inflammation. It is also aberrantly expressed in certain tumors, notably dermatofibrosarcoma protuberans (DFSP), a cutaneous characterized by strong CD34 positivity in tumor cells. CD34 expression patterns are conserved across species, with the murine ortholog showing similar distribution in endothelial and stromal cells. However, differences exist, particularly in the , where human CD34 is selectively expressed in endothelial cells, while murine expression includes broader embryonic and contexts with distinct regulatory mechanisms.

Biological Functions

Cell Adhesion and Migration

CD34, a sialomucin expressed on the surface of endothelial cells in high endothelial venules (HEVs), serves as a primary ligand for (CD62L) on circulating leukocytes, enabling initial tethering and rolling along the vascular wall during and immune surveillance. This interaction is crucial for leukocyte homing to lymph nodes and sites of , where the sulfated and sialylated glycoforms of CD34 present on HEVs bind with high affinity, facilitating the capture of lymphocytes and neutrophils from blood flow under . In binding assays have demonstrated that recombinant CD34 or HEV extracts coated with CD34 support -dependent rolling of lymphocytes, confirming its role in the initial phase of leukocyte recruitment. In addition to its pro-adhesive function, sialylated CD34 exhibits an inhibitory role in cell by virtue of its heavily glycosylated extracellular domain, which carries a dense array of negatively charged sialic acids. This negative charge creates a repulsive electrostatic barrier that hinders close apposition of cells and repels positively charged domains on , thereby preventing premature or non-specific firm and promoting a regulated migratory . Overexpression of CD34 in cell lines has been shown to reduce to extracellular matrix components and induce cell rounding, underscoring its anti-adhesive properties in modulating cell-cell and cell-matrix interactions. CD34 on HEV endothelium further contributes to transendothelial migration by supporting the transition from rolling to diapedesis for neutrophils and T-cells. As a component of the peripheral node addressin (PNAd) complex, CD34 enables the localized presentation of ligands that guide paracellular or transcellular passage of leukocytes through the endothelial barrier during immune responses. Experimental evidence from CD34 knockout mouse models reveals impaired leukocyte trafficking, including delayed neutrophil and eosinophil recruitment to sites of inflammation such as the lung in response to bacterial endotoxin, highlighting CD34's necessity for efficient migration in vivo.

Roles in Hematopoiesis and Angiogenesis

CD34 contributes to hematopoiesis by promoting the and retention of hematopoietic stem cells (HSCs) within specialized niches, supporting their quiescence and long-term repopulating potential. These niches provide a supportive microenvironment that preserves the long-term repopulating potential of HSCs, preventing premature and exhaustion. In transplant models, CD34-positive cells demonstrate robust multilineage reconstitution capabilities, generating all major blood cell lineages including erythrocytes, leukocytes, and platelets. For instance, transplantation of purified CD34+ cells from sources like or mobilized peripheral blood into immunodeficient mice results in long-term engraftment and sustained hematopoiesis, confirming their identity. These findings underscore CD34 as a key marker for isolating HSCs capable of repopulating the hematopoietic system. Beyond hematopoiesis, CD34 contributes to , particularly through its expression on endothelial cells (EPCs), where it promotes in ischemic tissues. EPCs bearing CD34 migrate to sites of , incorporating into nascent vessels or secreting paracrine factors to stimulate endothelial growth and repair. This process is essential for restoring blood flow in conditions like . Recent studies as of 2024-2025 have highlighted CD34+ cells' efficacy in promoting for diabetic wound repair and modulating tumor via endothelial interactions. CD34 is also involved in vascular endothelial growth factor (VEGF)-mediated differentiation of progenitor cells into endothelial lineages, enhancing tube formation and vascular stability. Synergistic interactions between VEGF and components further amplify this differentiation, leading to mature endothelial phenotypes. The dual origin hypothesis posits that CD34-positive hemangioblasts serve as common precursors for both hematopoietic and endothelial lineages during embryogenesis. These bipotent cells, identified in early embryonic structures like the and aorta-gonad-mesonephros region, give rise to HSCs and endothelial cells through a shared developmental pathway. This concept is supported by differentiation of embryonic stem cells into blast colony-forming cells that co-express CD34 and generate both lineages. Recent studies from 2023 have refined the understanding of CD34's position in the hierarchy, revealing that CD34+CD38–CD90+CD45RA– subsets are highly enriched for long-term repopulating HSCs, with CD34-negative populations potentially representing even more primitive progenitors. Additionally, investigations into EPC-based therapies have demonstrated improved efficacy of CD34+ cell infusions in treating ischemic diseases, such as enhanced vascular repair in models through paracrine mechanisms.

Clinical Applications

Stem Cell Transplantation

CD34 serves as a key marker for the and of hematopoietic stem and progenitor cells (HSPCs) in transplantation, enabling the purification of viable grafts for therapeutic reconstitution of the hematopoietic system following myeloablative conditioning in conditions such as and . In autologous and allogeneic (HSCT), the CD34+ cell dose is a critical determinant of successful engraftment, with doses exceeding 2 × 10^6 CD34+ cells per kilogram of recipient body weight associated with timely and platelet recovery, typically within 10-14 days post-transplant. Higher doses, such as greater than 5 × 10^6/kg, further accelerate engraftment kinetics and improve overall outcomes in patients undergoing autologous HSCT. This predictive value stems from CD34's expression on long-term repopulating HSPCs, allowing pre-transplant assessment to guide graft processing and infusion. Isolation of CD34+ cells for transplantation primarily employs immunomagnetic selection or fluorescence-activated cell sorting (FACS) to achieve high purity and viability. Immunomagnetic methods use antibody-coated beads to deplete non-CD34+ cells or positively select CD34+ populations from mobilized peripheral blood or bone marrow mononuclear cells, yielding recoveries of 70-90% with purities often exceeding 95%. For enhanced stem cell enrichment, flow cytometry sorting targets CD34+ CD38- subsets, which represent primitive HSPCs with superior long-term repopulating potential, minimizing mature cell contamination and improving graft potency. These techniques, validated in clinical protocols, facilitate the removal of T cells to mitigate graft-versus-host disease (GVHD) while preserving engraftment capacity. The clinical application of CD34-guided transplantation emerged in the , with early studies demonstrating rapid engraftment and reduced GVHD incidence using purified CD34+ cells from G-CSF-mobilized peripheral blood in allogeneic settings. A seminal trial reported successful reconstitution in 12 high-risk patients infused with a median of 2.9 × 10^6 CD34+ cells/kg, achieving engraftment by median day 14 without acute GVHD in most cases, highlighting the approach's efficacy in T-cell depleted grafts. Subsequent outcomes confirmed that CD34+ selection lowers severe GVHD rates to below 20% compared to unmanipulated grafts, though at the potential cost of attenuated graft-versus- effects in some malignancies. Low CD34+ cell doses below 2 × 10^6/kg are linked to delayed or failed engraftment, increasing risks of , hemorrhage, and transplant-related mortality in both autologous and allogeneic HSCT for hematologic cancers. Post-transplant via chimerism assays, which quantify donor versus recipient proportions through PCR-based of short tandem repeats or SNPs, enables early detection of engraftment status and guides interventions like booster infusions. Complete donor chimerism by day 14 correlates with robust myeloid recovery, while mixed or low donor chimerism signals potential complications requiring prompt evaluation.

Regenerative and Diagnostic Uses

CD34+ endothelial cells (EPCs) have shown promise in regenerative therapies for vascular diseases, particularly through their angiogenic properties that promote repair. In critical limb ischemia (CLI), a severe form of , the phase 3 SALAMANDER trial (initiated in 2017) evaluated autologous CD34+-enriched mononuclear cell preparations, such as REX-001, but was terminated in 2022 due to futility, with no demonstrated improvement in or reduced rates in patients with and Rutherford category 5 CLI as of 2025. Emerging applications extend to other ischemic conditions. In liver cirrhosis, a 2024 phase II clinical trial demonstrated that hepatic arterial infusion of autologous peripheral blood-derived CD34+ cells improved liver function scores and delayed decompensation in decompensated patients, potentially averting the need for transplantation through enhanced vascularization and hepatocyte support. In diagnostics, CD34 serves as a key immunohistochemical marker for distinguishing certain tumors. Solitary fibrous tumors consistently express CD34 in over 90% of cases, aiding in their identification among spindle cell neoplasms, whereas is typically CD34-negative, helping to rule out this entity in differential diagnoses. utilizing CD34 enables sensitive detection of (MRD) in (AML), with MRD levels below 0.1% post-induction correlating with better relapse-free survival in multiparametric assays. CD34+ subsets also mark therapy-resistant cancer stem cells (CSCs) in various malignancies. In AML, CD34+CD38- populations represent leukemia-initiating cells that confer resistance to , as confirmed in 2023 analyses linking their persistence to higher relapse rates.

Interactions and Regulation

Protein Interactions

CD34, a sialomucin expressed on hematopoietic / cells and endothelial cells, engages in several key protein interactions that mediate and signaling. One primary extracellular interaction occurs between CD34 and (CD62L), where L-selectin on leukocytes binds to specific glycoforms of CD34 on high endothelial venules, facilitating initial tethering and rolling during . This binding is dependent on sulfated (6-sulfo sLe^x) moieties on both O- and N-glycans of CD34, with only a minor subset (<10%) of CD34 glycoforms exhibiting functional ligand activity. Experimental confirmation of this interaction has been achieved through , which isolates -competent CD34 glycoforms, and assays demonstrating leukocyte adhesion to CD34-transfected cells. Intracellularly, the cytoplasmic tail of CD34 associates with the adapter protein CRKL, a 39-kDa molecule containing SH2 and SH3 domains that links receptor kinases to downstream effectors. This association occurs via the membrane-proximal region of CD34 (sequence RRSWSPTGER) and CRKL's C-terminal SH3 domain, promoting cytoskeletal reorganization and signaling for and . Co-immunoprecipitation experiments from CD34-expressing KG1a cells and GST-pull down assays using CD34 intracellular domain fusion proteins have verified this direct binding, with no similar interactions observed for related adapters like CrkII or Grb2. Additional interactions involve galectin-1 and , which bind to moieties on CD34, modulating properties without direct protein-protein contact. Recombinant galectin-1 binds to CD34-positive blood cells, enhancing clonogenic potential in a -dependent manner inhibitable by thiodigalactoside, as shown by . Similarly, exogenous strongly binds CD34+ early myeloid cells, promoting G-CSF-driven proliferation, with Western blotting detecting galectin-3-associated proteins in these fractions. such as (α4β1) interact indirectly with CD34 through niche effects, where CD34 influences integrin-mediated during homing, though no direct binding has been established. These interactions yield functional outcomes in and dynamics. The CD34- regulates inflammatory leukocyte recruitment. CD34-CRKL association supports mobilization by integrating signals for cytoskeletal changes essential for egress from the niche. bindings further tune adhesion, preventing excessive retention in the marrow. affinities have been characterized, with L-selectin to sulfated CD34 glycoforms showing a (K_d) of approximately 47 μM, measured via analogous calibration in assays, indicative of moderate enhanced by multivalency. Co-immunoprecipitation and pull-down studies for CRKL confirm stable intracellular complexes, while carbohydrate inhibition assays validate interactions.

Gene Regulation

The regulation of CD34 gene expression is primarily governed by specific promoter elements that facilitate (HSC)-specific transcription. The CD34 promoter contains binding sites for key transcription factors, including RUNX1, which mediates interactions with a distal regulatory element to drive expression in HSCs. Additionally, SCL/TAL1 and GATA family factors, such as , bind to these regulatory regions, often in complex with other factors like FLI1, ERG, LYL1, and LMO2, to promote CD34 transcription in CD34+ HSPCs. These interactions ensure restricted expression in primitive hematopoietic progenitors. Epigenetic modifications further control CD34 availability by modulating accessibility. In hematopoietic , the CD34 promoter exhibits active marks, including high levels of , which correlate with open and transcriptional activation. Conversely, during , at the CD34 promoter increases significantly, leading to and loss of CD34 expression in mature cells. This shift is programmed early at the progenitor stage, where patterns establish stable repression in differentiated lineages. Several signaling pathways influence CD34 transcription through these regulatory elements. Canonical Wnt/β-catenin signaling upregulates CD34 expression by enhancing the frequency and maintenance of CD34+ cells in coculture systems with Wnt ligands. Similarly, signaling promotes CD34+ progenitor expansion and sustains expression by inhibiting , as seen in cultures with Notch ligands like Delta-1. Cytokines such as (SCF), when combined with others like IL-3 and G-CSF during protocols, contribute to downregulation of CD34 by driving commitment and loss of stemness. In pathological contexts, dysregulation of these mechanisms alters CD34 levels, particularly in (AML). Promoter hypermethylation in AML cells can reduce CD34 expression by silencing the , contributing to aberrant expansion. Studies highlight epigenetic therapies, such as hypomethylating agents, that target these alterations to restore normal regulation and improve outcomes in AML.

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