CCL28 Human, His

What is CCL28 and what are its primary biological functions?

CCL28 (Chemokine C-C motif ligand 28) is an immunoregulatory chemokine that functions as both a chemoattractant and, more recently discovered, a growth and survival factor for hematopoietic stem and progenitor cells (HSPCs). The protein is primarily expressed in mucosal tissues such as exocrine glands, trachea, and colon, particularly in tissues that secrete low-salt fluids . CCL28 signals through two cell surface receptors, CCR3 and CCR10, with CCR10 being predominantly expressed in primitive hematopoietic cells . Beyond its chemotactic properties, CCL28 directly stimulates proliferation of primitive hematopoietic cells from different ontogenetic origins, enhances functional progenitor cell content by stimulating cell cycling, and induces gene expression changes associated with survival . Additionally, CCL28 exhibits antimicrobial activity, suggesting a dual role in mucosal immunity .

How does the His-tagged recombinant version of CCL28 differ from native CCL28?

The His-tagged recombinant version of human CCL28 contains a polyhistidine tag (typically 6×His) that is genetically engineered onto the protein, usually at the N- or C-terminus. While the core protein maintains the same amino acid sequence as native CCL28, the addition of the His-tag facilitates protein purification through immobilized metal affinity chromatography (IMAC). Research shows that properly produced His-tagged CCL28 maintains its biological activities, as demonstrated in studies where it successfully promoted expansion of CD34+ hematopoietic cells at concentrations between 50-1000 ng/mL, with effects reaching saturation at approximately 500 ng/mL . When using His-tagged CCL28 in experimental settings, researchers should validate that the tag does not interfere with receptor binding or downstream signaling pathways by comparing its activity to the untagged version in functional assays.

Which receptors does CCL28 interact with and how do they mediate its functions?

CCL28 signals through two distinct G protein-coupled receptors: CCR3 and CCR10 . Receptor expression analysis of hematopoietic stem and progenitor cells reveals that CCR10 is expressed in the vast majority of CD34+ cells, including the HSC-enriched CD34+CD38- population, while CCR3 expression is largely absent in these primitive populations . This expression pattern suggests that CCL28 primarily operates through CCR10 on the earliest hematopoietic progenitors. The CCL28-CCR10 signaling axis appears crucial for mediating CCL28's growth-promoting and survival effects on HSPCs, as evidenced by increased cell numbers and progenitor activity in CCL28-treated cultures compared to those with SCF alone . The receptor engagement triggers downstream pathways that suppress apoptosis and stimulate cell cycling, thereby supporting the proliferation and maintenance of hematopoietic progenitors .

What are the optimal culture conditions for studying CCL28 effects on hematopoietic stem cells?

For optimal study of CCL28 effects on hematopoietic stem cells, researchers should use serum-free expansion medium supplemented with stem cell factor (SCF) at 10 ng/mL (referred to as "S10") as a baseline condition . CCL28 demonstrates dose-dependent effects from 50-1000 ng/mL, with 500 ng/mL being the optimal concentration where effects reach saturation . When designing experiments, consider the following parameters:

For assessing CCL28 effects in different cytokine contexts, it can be combined with other factors like thrombopoietin (TPO) or fms-like tyrosine kinase 3 ligand (FLT3L), though CCL28 appears to synergize most effectively with SCF .

How should researchers quantify and evaluate CCL28-mediated expansion of hematopoietic cells?

To comprehensively evaluate CCL28-mediated expansion of hematopoietic cells, researchers should implement a multi-parameter assessment approach:

• Proliferation Assessment: Track total cell numbers and fold expansion using standard cell counting techniques. Flow cytometry using CD34 expression helps distinguish primitive progenitors from more differentiated cells .

• Progenitor Cell Frequency: Perform colony-forming cell (CFC) assays according to manufacturer's protocols (e.g., StemCell Technologies) to quantify functional progenitors. Different colony types (CFU-GM, BFU-E, CFU-GEMM) should be enumerated separately to assess lineage distribution .

• Long-Term Culture-Initiating Cell (LTC-IC) Assays: Establish these to evaluate more primitive progenitors that aren't detected in standard CFC assays. LTC-IC frequency should be compared between treatment conditions and relative to fresh cell equivalents .

• Phenotypic Analysis: Use flow cytometry to analyze marker combinations including CD34, CD38, CD90, and CD45RA to identify putative HSCs (CD34^hi CD38^lo CD90^+ CD45RA^-) and track their maintenance in culture .

• In Vivo Repopulation: For definitive assessment of HSC activity, transplant cultured cells into immunodeficient mice (e.g., NSG) and evaluate multi-lineage engraftment at both early (4 weeks) and late (16+ weeks) timepoints to distinguish between short-term and long-term repopulating cells .

Researchers should compare CCL28-treated cells not only to other cytokine conditions but also to equivalent numbers of fresh cells to determine whether CCL28 maintains or expands functional HSC activity .

What experimental controls are essential when investigating CCL28 effects on hematopoietic cells?

When investigating CCL28 effects on hematopoietic cells, the following controls are essential:

• Cytokine-Free Control: Cells cultured without any cytokines to establish baseline survival and differentiation rates.

• SCF-Only Control: Cells cultured with SCF alone (typically at 10 ng/mL) to distinguish CCL28-specific effects from those of the baseline growth factor .

• TPO Positive Control: Cells cultured with SCF+TPO, as TPO is an established HSC-supportive factor that provides a reference point for CCL28's efficacy .

• Fresh Cell Equivalent Control: Uncultured cells analyzed in parallel functional assays (CFCs, LTC-ICs, transplantation) to determine whether cultured cells maintain, lose, or exceed the functional capacity of the input population .

• Receptor Blocking Control: Where possible, include conditions with CCR10-blocking antibodies to confirm receptor specificity of observed effects.

• Dose Titration: Multiple concentrations of CCL28 (e.g., 50, 100, 500, 1000 ng/mL) to establish dose-response relationships .

• Time Course Analysis: Evaluation at multiple timepoints to distinguish between temporary and sustained effects of CCL28 treatment.

These controls help isolate CCL28-specific effects and provide appropriate context for interpreting experimental results across different functional assays.

How does CCL28 function as both a chemokine and a growth factor for HSPCs?

Gene expression analysis of CCL28-treated hematopoietic cells reveals upregulation of genes associated with cellular survival and downregulation of pro-apoptotic factors . This suggests that CCL28 activates anti-apoptotic pathways, which is critical for maintaining HSPC viability during ex vivo culture. Additionally, CCL28 stimulates cell cycling, as evidenced by increased proliferation of primitive hematopoietic cells from multiple ontogenetic origins .

The molecular bridge between chemokine and growth factor functions might involve shared signaling nodes downstream of CCR10 activation. The predominant expression of CCR10 on CD34+ cells, particularly within the HSC-enriched CD34+CD38- population, suggests that this receptor mediates both functions in primitive hematopoietic cells . Further research using phosphoproteomic approaches would help elucidate the specific signaling pathways that differentiate CCL28's chemotactic versus growth-promoting activities.

What are the optimal experimental designs for studying CCL28 effects on long-term hematopoietic stem cell repopulation?

Designing rigorous experiments to study CCL28 effects on long-term hematopoietic stem cell repopulation requires careful consideration of multiple parameters:

• Cell Population Selection: Start with highly purified HSCs (CD34^hi CD38^lo CD90^+ CD45RA^-) rather than bulk CD34+ cells to eliminate confounding effects from progenitor expansion .

• Limiting Dilution Analysis (LDA): Transplant varying cell doses to calculate HSC frequency rather than relying solely on fixed-cell-number transplants. This approach provides quantitative measurement of functional HSC maintenance/expansion.

• Competitive Transplantation: Include a competitive transplant design where CCL28-treated cells are transplanted alongside a standard competitor population (different fluorescent marker or CD45 allele) to directly compare repopulating ability.

• Serial Transplantation: Perform secondary and tertiary transplants using bone marrow from primary recipients to assess self-renewal capacity and long-term HSC maintenance.

• Multi-Lineage Analysis: Comprehensively analyze engraftment across all lineages (myeloid, B-lymphoid, T-lymphoid) at multiple timepoints, as short-term repopulating cells may show lineage-biased engraftment patterns.

• Molecular Barcode Tracking: Consider using cellular barcoding techniques to track individual HSC clones and their progeny, providing insights into clonal dynamics and potential selection effects of CCL28.

• Time Course Assessments: Evaluate engraftment at both early (4 weeks) and late (16+ weeks) timepoints to distinguish between effects on short-term versus long-term repopulating cells .

Research has shown that CCL28 treatment significantly improved long-term multilineage reconstitution levels compared to both fresh cells and cells cultured with SCF alone, demonstrating that the combined effect of SCF and CCL28 supports a net increase of long-term repopulating activity .

How does CCL28 interact with other cytokines in hematopoietic stem cell maintenance and expansion?

CCL28 demonstrates complex interaction patterns with other cytokines in hematopoietic stem cell maintenance and expansion. The following patterns have been observed:

• Synergy with SCF: CCL28 shows robust synergistic effects with stem cell factor (SCF), particularly at low SCF concentrations (10 ng/mL). This combination significantly enhances CD34+ cell expansion and functional progenitor content compared to either factor alone .

• Partial Synergy with TPO and FLT3L: CCL28 promotes growth in combination with thrombopoietin (TPO) or fms-like tyrosine kinase 3 ligand (FLT3L), but to a lesser extent than with SCF .

• Context-Dependent Effects in Rich Cytokine Cocktails: When added to a combination of SCF, TPO, and FLT3L (STF), CCL28 enhances the output of early progenitors with mixed CFC potential but does not rescue the adverse effects of STF stimulation on NSG reconstitution ability . This suggests that cytokine-rich environments may drive proliferation at the expense of maintaining primitive HSC properties.

• Minimal Activity as Single Agent: When added alone, CCL28 fails to induce net proliferation of CD34+ cells but maintains progenitor activity, suggesting it primarily provides crucial survival signals for primitive hematopoietic cells in the absence of other factors .

These interaction patterns highlight the context-dependent function of CCL28 and support the notion that optimal ex vivo culture conditions for HSPCs should be designed with careful consideration of cytokine combinations. Simple cytokine cocktails (e.g., SCF+CCL28) may better preserve HSC properties than complex mixtures that drive extensive proliferation .

How can researchers resolve discrepancies between in vitro and in vivo effects of CCL28 on hematopoietic stem cells?

Discrepancies between in vitro and in vivo effects of CCL28 on hematopoietic stem cells can arise from multiple sources. Researchers should systematically address these using the following approaches:

• Reconsider Cell Purity: Ensure experiments use well-defined HSPC populations. Bulk CD34+ populations contain heterogeneous progenitors that may respond differently to CCL28 than purified HSCs (CD34^hi CD38^lo CD90^+ CD45RA^-) . In vitro assays may inadvertently favor expansion of more committed progenitors that lack long-term engraftment potential.

• Evaluate Dose-Dependent Effects: CCL28 demonstrates dose-dependent effects from 50-1000 ng/mL in vitro . Inappropriate concentrations in either system could explain contradictory outcomes. Conduct comprehensive dose-response studies in both settings.

• Assess Receptor Expression Dynamics: Monitor CCR10 expression throughout culture period, as receptor downregulation could contribute to diminished responsiveness over time. Flow cytometry analysis of receptor expression before and after culture provides insight into potential desensitization mechanisms.

• Consider Cytokine Combinations Carefully: CCL28 effects depend significantly on co-stimulatory factors. While it synergizes with SCF to maintain HSC activity, this effect is lost in rich cytokine cocktails (SCF+TPO+FLT3L) . Match in vitro cytokine conditions with the physiological context of transplantation models.

• Examine Engraftment Kinetics: CCL28-treated cells may show different engraftment kinetics compared to fresh cells. Research suggests potentially delayed engraftment of cultured cells, necessitating extended monitoring periods in transplantation studies .

• Use Multiple Functional Readouts: Correlate in vitro surrogate assays (CFC, LTC-IC) with in vivo transplantation outcomes to identify which in vitro metrics best predict in vivo performance of CCL28-treated cells .

By systematically addressing these factors, researchers can better understand and reconcile discrepancies between different experimental systems, ultimately improving the translation of in vitro findings to in vivo applications.

What are common pitfalls in experimental design when studying CCL28 effects on hematopoietic cells?

When studying CCL28 effects on hematopoietic cells, researchers should be aware of several common pitfalls:

• Inappropriate Baseline Cytokine Conditions: Using high concentrations of SCF (100 ng/mL) or complex cytokine cocktails can mask the specific effects of CCL28. Research shows that CCL28's effects are best distinguished using low concentrations of SCF (10 ng/mL) that maintain survival without extensive proliferation .

• Insufficient Duration for HSC Assessment: Short-term culture periods may be adequate for progenitor analysis but insufficient for evaluating true HSC maintenance. Extended culture periods (10-14 days) may be necessary to reveal differences in HSC preservation between conditions.

• Reliance on Phenotypic Markers Alone: CD34 expression alone is insufficient to identify HSCs. Without additional markers (CD38, CD90, CD45RA) or functional assays, researchers may misinterpret expansion of committed progenitors as HSC expansion .

• Overlooking Receptor Expression Analysis: Failure to verify CCR10 expression on target cell populations may lead to inconclusive results, particularly when working with heterogeneous or differentiated cell types where receptor expression patterns may differ.

• Inadequate In Vivo Assessment: Transplantation studies with single timepoint analysis or without multi-lineage differentiation assessment provide incomplete information about HSC functionality. Research has shown that CCL28-treated cells may show different engraftment patterns at early versus late timepoints post-transplantation .

• Protein Quality Issues: Recombinant CCL28 quality, particularly with His-tagged variants, can vary between suppliers. Activity validation through dose-response proliferation assays should be performed with each new lot.

• Insufficient Statistical Power: Due to biological variability in primary hematopoietic cells, experiments should include multiple biological replicates (minimum n=3) from different donors to ensure reproducibility of observed CCL28 effects.

By avoiding these pitfalls, researchers can design more robust experiments that accurately characterize CCL28's effects on hematopoietic stem and progenitor cells.

How can researchers distinguish between CCL28's effects on proliferation versus survival of hematopoietic stem cells?

Distinguishing between CCL28's effects on proliferation versus survival of hematopoietic stem cells requires specific experimental approaches that separately quantify these cellular processes:

• Cell Cycle Analysis: Implement BrdU incorporation or Ki-67/DAPI staining followed by flow cytometry to assess cell cycle distribution. This approach reveals whether CCL28 increases the proportion of cells in S/G2/M phases, indicating enhanced proliferation. Research has shown that CCL28 stimulates cell cycling in primitive hematopoietic cells .

• Division Tracking: Use CellTrace Violet or CFSE labeling to track sequential cell divisions. Flow cytometry analysis of dye dilution provides quantitative data on proliferation rates and division history of individual cells.

• Apoptosis Assays: Employ Annexin V/7-AAD staining to quantify early and late apoptotic cells. Comparison between CCL28-treated and control conditions reveals survival-promoting effects. Gene expression studies indicate that CCL28 induces changes associated with enhanced survival .

• Single-Cell Survival Analysis: Use time-lapse microscopy with microwell arrays to track individual cell survival times, independent of proliferation effects.

• Gene Expression Profiling: Analyze expression of proliferation markers (e.g., cyclins, CDKs) versus survival factors (e.g., BCL2 family proteins) to identify which pathways are predominantly activated by CCL28. Previous transcriptional profiling has identified gene expression changes associated with survival in CCL28-treated cells .

• Sequential Factor Addition: Design experiments where CCL28 is added at different timepoints relative to other factors. Early effects on survival can be distinguished from later effects on proliferation.

• Inhibitor Studies: Use specific inhibitors of cell cycle progression (e.g., CDK inhibitors) versus apoptosis (e.g., caspase inhibitors) to determine which pathway predominantly mediates CCL28's effects.

The research indicates that CCL28 supports both mechanisms - it enhances proliferation by stimulating cell cycling while simultaneously inducing gene expression changes associated with survival . When added alone, CCL28 fails to induce net proliferation but maintains progenitor activity, suggesting a strong survival component to its function .

What are promising avenues for investigating CCL28's role in the bone marrow microenvironment?

Given the presence of CCL28 in the bone marrow microenvironment, several promising research avenues warrant investigation:

• Cellular Sources of CCL28: Identify which specific niche cells (mesenchymal stromal cells, endothelial cells, osteoblasts) produce CCL28 within the bone marrow using single-cell RNA sequencing and in situ hybridization techniques .

• Spatial Distribution Analysis: Map the spatial distribution of CCL28 protein relative to phenotypically defined HSCs using multiplexed immunofluorescence or CODEX imaging to determine whether CCL28 concentration gradients exist within distinct bone marrow niches.

• Stress-Induced Regulation: Investigate how physiological stresses (inflammation, hypoxia, irradiation, chemotherapy) modify CCL28 expression patterns in the bone marrow microenvironment and how these changes impact hematopoietic recovery.

• Genetic Models: Develop conditional knockout models targeting CCL28 or CCR10 in specific bone marrow niche populations to assess the physiological importance of this signaling axis for steady-state and stress hematopoiesis.

• Exogenous Administration: Evaluate whether systemic or local administration of recombinant CCL28 enhances hematopoietic recovery after myeloablative injury by supporting endogenous HSPC survival and proliferation.

• Niche Competition Studies: Determine whether CCL28 influences competition between normal HSCs and malignant cells for niche resources, potentially informing new therapeutic approaches for hematological malignancies .

• Cross-talk with Other Niche Factors: Explore how CCL28 interacts with established niche factors (CXCL12, SCF, TPO) to collectively regulate HSC maintenance and mobilization.

These research directions would significantly advance understanding of CCL28's physiological role in regulating hematopoiesis within the complex bone marrow microenvironment and potentially reveal new therapeutic applications for modulating HSPC function in vivo .

How might CCL28 be utilized in clinical applications for hematopoietic stem cell transplantation?

CCL28 shows promising potential for clinical applications in hematopoietic stem cell transplantation (HSCT) through several strategic approaches:

• Ex Vivo Expansion Protocols: Incorporate CCL28 (optimally at 500 ng/mL) alongside SCF in clinical-grade expansion protocols to enhance the functional HSC content of cultured grafts. Research demonstrates that CCL28 significantly improves long-term multilineage reconstitution capacity compared to equivalent input numbers of fresh cells .

• Cord Blood Enhancement: Apply CCL28 specifically to cord blood expansion protocols to address the limitation of low cell numbers in single cord blood units, potentially eliminating the need for double cord transplants.

• Engraftment Acceleration: Investigate whether cultures containing CCL28 might accelerate early neutrophil and platelet recovery in transplant recipients, reducing the duration of post-transplant cytopenia.

• Serum-Free Formulations: Develop GMP-compliant, defined media formulations containing recombinant human CCL28 for clinical application, avoiding xenogeneic components that complicate regulatory approval.

• In Vivo Administration: Explore direct administration of CCL28 to transplant recipients during the post-transplant period to support survival and expansion of infused HSCs, potentially in combination with other growth factors like G-CSF.

• Selective Expansion: Utilize CCL28's preferential effects on primitive hematopoietic cells to develop protocols that selectively expand HSCs while minimizing expansion of committed progenitors or mature cells that contribute to graft-versus-host disease.

• Gene-Modified Cell Products: Investigate whether CCL28 treatment might preferentially support gene-modified HSCs during ex vivo manipulation, potentially improving outcomes for gene therapy approaches.

Before clinical implementation, additional studies must address optimal timing, dosing, potential immunogenicity of recombinant CCL28, and comprehensive safety assessments. The research suggests that CCL28 may be particularly valuable in minimal cytokine cocktails that avoid the adverse effects of complex growth factor combinations on HSC maintenance .

What are the implications of CCL28's dual role in mucosal immunity and hematopoiesis for understanding tissue-specific regulation of stem cells?

The dual functionality of CCL28 in both mucosal immunity and hematopoiesis offers profound insights into tissue-specific regulation of stem cells:

• Evolutionary Conservation of Dual Functions: CCL28's antimicrobial and growth-promoting activities suggest evolutionary conservation of molecular pathways that simultaneously regulate host defense and tissue regeneration . This dual role may represent an efficient mechanism where a single molecule serves multiple purposes in maintaining tissue homeostasis.

• Microenvironmental Cross-Regulation: CCL28's expression in mucosal tissues that secrete low-salt fluids (salivary glands, colon, trachea) and the bone marrow suggests common regulatory mechanisms across distinctly different tissue microenvironments . This raises questions about how tissue-specific signals modify CCL28's function in different anatomical contexts.

• Inflammatory Signaling in Stem Cell Regulation: As an immunoregulatory chemokine with stem cell growth factor activity, CCL28 represents a molecular link between inflammatory signaling and stem cell maintenance. This connection may explain how inflammation influences stem cell behavior during tissue repair and regeneration.

• Compartmentalized Receptor Expression: The differential expression of CCL28 receptors (CCR10 vs. CCR3) in hematopoietic versus mucosal tissues provides a mechanism for context-specific responses to the same ligand . This principle may apply to other molecules with pleiotropic effects across multiple tissue systems.

• Niche Evolution Theory: CCL28's dual role supports theories proposing that specialized tissue niches evolved from more primitive defense mechanisms, with molecules originally serving antimicrobial functions being repurposed for sophisticated cellular regulation.

• Therapeutic Targeting Considerations: The multifunctional nature of CCL28 highlights the importance of considering tissue-specific effects when developing therapeutic approaches targeting chemokine pathways.

These implications suggest that further investigation of molecules with dual roles in immunity and stem cell regulation may reveal fundamental principles about the evolution of tissue-specific niches and provide novel therapeutic targets for both inflammatory disorders and regenerative medicine applications .

What is the current consensus on CCL28's significance in hematopoietic stem cell biology?

• Novel Growth Factor Status: CCL28 is firmly established as a potent growth-promoting factor with the ability to support the in vitro and in vivo functional properties of cultured human hematopoietic cells .

• Direct Action on Primitive Cells: Evidence clearly demonstrates that CCL28 directly stimulates proliferation of primitive hematopoietic cells through the CCR10 receptor, which is expressed on HSC-enriched CD34+CD38- populations .

• Dual Mechanistic Action: CCL28 enhances both proliferation (through cell cycle stimulation) and survival (through anti-apoptotic gene expression changes) of HSPCs, providing complementary support mechanisms .

• Contextual Efficacy: CCL28 functions optimally in combination with SCF, particularly at low SCF concentrations (10 ng/mL), but shows diminished benefit in complex cytokine cocktails that may drive differentiation at the expense of HSC maintenance .

• Transplantation Enhancement: Addition of CCL28 to cultures of purified putative HSCs significantly increases their ability to long-term repopulate immunodeficient mice compared to equivalent input numbers of fresh cells, demonstrating true functional enhancement of HSC activity .

• Physiological Relevance: The presence of CCL28 in the bone marrow microenvironment suggests it may have a physiological role in regulating HSPCs in vivo, though this requires further investigation .

How does our understanding of CCL28 contribute to broader concepts in stem cell biology?

Our understanding of CCL28 contributes significantly to broader concepts in stem cell biology through several important paradigms:

• Chemokine Signaling in Stem Cell Regulation: CCL28's role challenges the traditional view that chemokines primarily regulate cell migration, revealing that they can directly influence stem cell self-renewal and differentiation decisions. This expands our understanding of the molecular language governing stem cell behavior beyond classical growth factors .

• Microenvironmental Complexity: The dual function of CCL28 in different tissue contexts (mucosal immunity and hematopoiesis) highlights the complexity of microenvironmental regulation and suggests that tissue-specific stem cell niches may share molecular mechanisms across anatomically distinct locations .

• Minimalist Supportive Conditions: CCL28's ability to maintain HSC function with minimal cytokine support (primarily SCF) contributes to the concept that simpler conditions may better preserve stem cell properties than complex growth factor cocktails that drive extensive proliferation and differentiation .

• Evolutionary Conservation of Stem Cell Regulators: The preservation of CCL28's dual functionality in immunity and hematopoiesis suggests evolutionary conservation of molecular pathways regulating both defense and regeneration, providing insight into the developmental origins of specialized stem cell niches.

• Receptor Specificity in Stem Cell Populations: The finding that HSCs predominantly express CCR10 rather than CCR3 demonstrates how receptor expression patterns can dictate cell type-specific responses to widely expressed ligands, adding another layer of regulation to stem cell behavior .

• Translational Bridge Between In Vitro and In Vivo Function: CCL28's ability to enhance transplantable HSC activity illustrates the important principle that true stem cell expansion requires maintaining in vivo functionality, not merely phenotypic markers or in vitro proliferation .