OSM Human, 195 a.a

Which receptor systems does Human OSM utilize for signaling?

Human OSM exhibits unique dual receptor utilization capabilities:

• OSM can signal through a heterodimeric complex consisting of the OSM-specific receptor (OSMR) and the signal transducing subunit gp130 .

• Human OSM can also utilize the leukemia inhibitory factor receptor (LIFR) in combination with gp130 .

This dual receptor usage distinguishes human OSM from its mouse counterpart, which primarily signals through mouse OSMR with limited activity on mouse LIFR . The receptor binding is determined by specific regions including the AB loop and N-terminal helix D, which are critical for receptor recognition and activation .

What are the primary cellular sources of Human OSM?

Human OSM is primarily synthesized by:

• Stimulated T-cells and monocytes

• Macrophages, as identified through single-cell RNA sequencing from human lung biopsies

These immune cells release OSM in response to various stimuli, particularly in inflammatory conditions. For example, bacterial lipopolysaccharide (LPS) has been demonstrated to induce OSM expression in macrophages .

How is Human OSM (195 a.a.) utilized in stem cell research protocols?

Human OSM plays a crucial role in stem cell differentiation protocols, particularly:

• Differentiation of human pluripotent stem cells into hepatocyte-like cells

• Regulation of the hematopoietic stem cell niche in the bone marrow

• Supporting osteogenesis and neurogenesis processes

For optimal results in differentiation protocols, OSM is typically used in combination with other growth factors and cytokines. Common complementary factors include:

These combinations enhance the efficiency and specificity of directed differentiation processes .

What methodologies exist for assessing Human OSM bioactivity?

Two validated methodologies for assessing Human OSM bioactivity include:

• Serum Response Element (SRE) Luciferase Reporter Assay

  • System: Transfected HEK293T cells

  • Protocol: Cells are treated in triplicate with serial dilutions of OSM for 6 hours

  • Measurement: Firefly luciferase activity normalized to control Renilla luciferase activity

  • Sensitivity: EC50 = 0.75 ng/ml (34 pM)

• Cell Proliferation Assay

  • System: Human TF-1 cells

  • Measurement: Dose-dependent proliferation response

  • Sensitivity: ED50 < 2 ng/ml

  • Activity correspondence: >5.0 × 10^5 IU/mg

These functional assays provide quantitative measures of OSM activity through either signaling pathway activation or cellular proliferation responses.

What are the optimal handling and storage conditions for maintaining Human OSM activity?

To maintain optimal activity of recombinant Human OSM (195 a.a.):

• Storage Form: Typically supplied as lyophilized powder from a 0.2μm filtered concentrated solution in PBS, pH 7.4

• Long-term Storage:

  • Temperature: -20°C to -70°C

  • Duration: Stable for up to 12 months from date of receipt when properly stored

• Working Solution Preparation:

  • Reconstitute in sterile buffer

  • For dilute solutions, addition of carrier proteins like BSA may prevent adsorption to tubes

  • Minimize freeze-thaw cycles by preparing single-use aliquots

• Quality Control Parameters:

  • Endotoxin content should be <0.1 EU/μg as determined by LAL method

  • Verify activity using bioassays before use in critical experiments

What molecular differences exist between Human and Mouse OSM relevant to experimental design?

Understanding the differences between Human and Mouse OSM is critical for experimental design, particularly when translating between in vitro human studies and mouse models:

Despite these differences, human OSM has demonstrated some activity on mouse ES cells, suggesting partial cross-species functionality in specific contexts .

Which specific amino acid residues determine species-specific receptor activation?

Site-directed mutagenesis experiments have identified specific amino acid residues within the AB loop that determine species-specific activities:

• Key residues in mouse OSM: Asn-37, Thr-40, and Asp-42 in the AB loop determine limited activation of mouse LIFR and human receptor activity

• Critical substitution: The Asp to Lys exchange is particularly important for evading mouse OSMR signaling by human OSM

• Structural basis: These amino acid differences alter the spatial configuration of binding site III, which is critical for receptor interaction

These findings highlight how minimal amino acid changes can dramatically alter cytokine-receptor specificity and provide mechanistic insights into the evolutionary divergence of OSM function between species.

How can chimeric OSM proteins be designed and utilized for cross-species research?

Chimeric OSM proteins provide valuable tools for cross-species research and have been successfully developed using homology modeling approaches:

Design Strategy:

• Generate a homology model based on the published human OSM structure (Protein Data Bank code 1EVS)

• Identify key regions for receptor specificity (AB loop and N-terminal helix D)

• Select appropriate replacement lengths for each region based on sequence alignment

• Introduce the human AB loop into mouse OSM or vice versa

Experimental Outcomes:

• Introduction of human AB loop in mouse OSM enables human OSMR and LIFR activation

• Presence of mouse loop in human OSM facilitates mouse OSMR signaling

Research Applications:

• Creating OSM variants with predictable receptor activation profiles

• Developing mouse models with humanized OSM signaling

• Studying receptor-specific biological responses across species

• Facilitating preclinical studies in mice using OSM variants with human-like functional features

How does Human OSM contribute to inflammatory disease pathophysiology?

Human OSM plays significant roles in inflammatory disease pathophysiology, particularly in severe asthma:

Pathophysiological Mechanisms:

• Bacterial Trigger Response: Bacterial lipopolysaccharide (LPS) induces OSM expression

• Cellular Source: Macrophages produce OSM in response to bacterial stimuli

• Signal Translation: Macrophage-derived OSM translates LPS signals into asthma-associated pathologies

• Clinical Correlation: Airway biopsies from patients with severe asthma present with an OSM-related gene signature

Experimental Evidence:

• Blockade of OSM with an OSM-specific antibody reduced severe asthma-related symptoms in mice after exposure to bacterial stimuli

• Studies using Osm-deficient murine macrophages demonstrated the essential role of macrophage-derived OSM in pathology development

These findings establish OSM as a critical mediator in the pathway from bacterial exposure to inflammatory airway disease, with significant therapeutic implications.

What methodological approaches are optimal for studying OSM signaling in disease models?

Multiple complementary methodological approaches have proven effective for studying OSM signaling in disease models:

• Genetic Approaches:

  • Gene knockout models (Osm-deficient mice)

  • CRISPR/Cas9-mediated gene editing

  • siRNA-mediated knockdown in cellular systems

• Molecular Profiling:

  • Single-cell RNA sequencing to identify cellular sources of OSM

  • Transcriptomic analysis to characterize OSM-related gene signatures

  • Proteomics to map signaling cascade components

• Pharmacological Interventions:

  • Antibody-mediated OSM blockade in vivo

  • Receptor antagonists

  • Small molecule inhibitors of downstream signaling

• Functional Readouts:

  • Pathway activation assays (luciferase reporters)

  • Cellular response measurements (proliferation, differentiation)

  • Disease-specific phenotypic assessments (e.g., airway inflammation)

Combining these approaches allows for comprehensive investigation of OSM's role in disease pathophysiology from molecular mechanisms to systemic effects.

What therapeutic targeting strategies exist for OSM signaling pathways?

Based on current research, several strategies for therapeutic targeting of OSM signaling have been identified:

• Direct OSM Neutralization:

  • Anti-OSM monoclonal antibodies have demonstrated efficacy in reducing severe asthma-related symptoms in mouse models

  • Humanized antibodies targeting OSM are in development for clinical applications

• Receptor-Based Approaches:

  • OSM receptor (OSMR) antagonists

  • Soluble receptor decoys to sequester free OSM

  • Targeting the critical AB loop interactions with small molecules

• Downstream Signaling Inhibition:

  • JAK/STAT pathway inhibitors

  • MAPK pathway modulators

  • Targeting transcriptional regulators induced by OSM

• Source-Directed Strategies:

  • Targeting macrophage activation to prevent OSM production

  • Inhibiting the bacterial triggers (e.g., LPS) that induce OSM expression

The recognition that "inhibiting OSM [may] prevent bacterial-associated progression and exacerbation of severe asthma" provides strong rationale for further development of these therapeutic approaches.

What factors contribute to variable reproducibility in Human OSM experiments?

Several factors can affect the reproducibility of Human OSM experiments:

• Protein Quality Issues:

  • Purity variations (optimal preparations should be >97% pure by SDS-PAGE and HPLC)

  • Endotoxin contamination (should be <0.1 EU/μg)

  • Conformational stability affected by storage and handling

• Experimental Design Factors:

  • Receptor expression levels in target cells

  • Species-specific activity considerations when using human OSM in non-human systems

  • Presence of antagonistic factors in culture media

• Technical Considerations:

  • Dosage optimization (effective concentration range: 0.75-2 ng/ml)

  • Timing of OSM addition in differentiation protocols

  • Co-factor requirements in specific cell types

• Readout Selection:

  • Sensitivity of assay systems (luciferase vs. proliferation)

  • Temporal dynamics of response measurements

  • Appropriate positive and negative controls

Understanding and controlling these variables is essential for achieving reproducible results in OSM research applications.

How can researchers optimize experimental conditions for Human OSM activity?

To optimize experimental conditions for Human OSM activity:

• Receptor Expression Verification:

  • Confirm target cells express appropriate receptors (OSMR/LIFR and gp130)

  • Consider receptor upregulation pretreatment if expression is low

• Dosage Titration:

  • Determine cell-specific dose-response curves

  • For most applications, start with concentration range of 0.5-5 ng/ml

  • EC50 values: ~0.75 ng/ml in SRE luciferase assay; <2 ng/ml in TF-1 proliferation assay

• Cofactor Optimization:

  • When using OSM for stem cell differentiation, test combinations with complementary factors (FGF-2, HGF NK1, activin A, TGF-β1)

  • Determine optimal sequential addition timing

• Experimental Readout Selection:

  • For signaling pathway activation: luciferase reporter assays

  • For functional responses: cell-type specific phenotypic assays

  • For molecular responses: phospho-specific antibodies for downstream mediators

• Quality Control:

  • Verify OSM bioactivity before critical experiments

  • Include appropriate positive controls

  • Use carrier proteins (e.g., BSA) for dilute working solutions

What alternative approaches can be employed when Human OSM experiments fail?

When Human OSM experiments fail, several alternative approaches can be considered:

• Alternative OSM Formulations:

  • Test different commercial sources of recombinant Human OSM

  • Consider chimeric OSM proteins with enhanced activity in your experimental system

  • Utilize OSM mutants with enhanced receptor specificity

• Related Cytokine Substitution:

  • Leukemia inhibitory factor (LIF), which is closely related to OSM

  • Other IL-6 family cytokines that activate overlapping signaling pathways

  • Engineered receptor-specific cytokine variants

• Receptor-Level Interventions:

  • Direct activation of downstream signaling components

  • Receptor overexpression to enhance sensitivity

  • Removal of potential inhibitory factors

• System Modifications:

  • Alternative cell types with higher receptor expression

  • Adjustment of culture conditions (serum levels, media formulation)

  • Preconditioning cells to enhance responsiveness

• Readout Adaptations:

  • More sensitive detection methods

  • Alternative endpoints that reflect the same biological process

  • Longer timepoints to capture delayed responses

By systematically working through these alternatives, researchers can overcome technical challenges and advance their OSM-related investigations.