PROCR Human

What is PROCR and what is its significance in human developmental biology?

PROCR (Protein C Receptor) is a cell surface receptor that has gained significant attention in developmental biology due to its potential role as a marker for progenitor cell populations. In human developmental contexts, PROCR expression has been identified in various tissues, with notable research focusing on its presence in pancreatic tissue development . The significance of PROCR lies in its association with cells that display both epithelial and mesenchymal characteristics, suggesting a potential role in cellular plasticity during development . Recent research has shown that PROCR+ cells in embryonic pancreatic tissue share transcriptional profiles with mesothelial cells, indicating their possible involvement in organ development and potentially in regenerative processes .

How can researchers reliably identify PROCR-expressing cells in human tissue samples?

Identification of PROCR-expressing cells in human tissue samples requires a multi-modal approach:

• Single-cell RNA sequencing (scRNA-seq): This technique allows for comprehensive transcriptional profiling of individual cells, enabling the identification of PROCR expression alongside co-expressed markers .

• Immunohistochemistry/Immunofluorescence: Using validated antibodies against PROCR and potential co-markers to spatially locate expressing cells within tissue architecture.

• Flow cytometry: For quantitative assessment of PROCR-expressing cells in dissociated tissues.

• Single-cell ATAC-seq: To assess the chromatin accessibility landscape of PROCR-expressing cells, which provides insights into the regulatory mechanisms controlling PROCR expression .

For reliable identification, researchers should consider using at least two complementary methods and include appropriate controls to validate specificity of detection methods .

What are the key differences between PROCR expression in human versus mouse pancreatic tissues?

Based on comparative analyses of mouse and human pancreatic samples, several key differences in PROCR expression patterns have been identified:

Importantly, while PROCR+ progenitor cells were initially reported in adult mouse pancreatic islets, subsequent studies have not consistently reproduced these findings in either mouse or human adult pancreatic tissue . Instead, cells with a similar transcriptional profile appear to be primarily associated with the mesothelial layer of the developing pancreas in both species .

What experimental models are most appropriate for studying PROCR function in human contexts?

For studying PROCR function in human contexts, researchers can employ several experimental models:

• Human pluripotent stem cell (hPSC) differentiation: Directing hPSCs toward pancreatic lineages can generate PROCR-expressing cells with similar transcriptional profiles to those found in embryonic development . This model allows for manipulation of PROCR expression and assessment of functional consequences.

• Organoid cultures: Three-dimensional organoid cultures derived from human embryonic pancreatic tissue or differentiated from hPSCs provide physiologically relevant models for studying PROCR+ cell behavior in a tissue-like environment .

• Xenotransplantation models: Human PROCR+ cells can be isolated and transplanted into immunodeficient mice to assess their developmental potential in vivo.

• CRISPR/Cas9-mediated genome editing: For functional studies of PROCR in human cell lines or stem cells, enabling precise manipulation of the gene and assessment of downstream effects.

When designing experiments using these models, researchers should implement a randomized block design to account for variability between cell lines or donor tissues .

How should researchers design experiments to investigate the potential progenitor function of PROCR+ cells in human pancreatic development?

Designing rigorous experiments to investigate the progenitor potential of PROCR+ cells requires careful consideration of several methodological aspects:

• Lineage tracing strategy:

  • In human pluripotent stem cell models, implement inducible genetic labeling systems (e.g., Cre-loxP) driven by the PROCR promoter

  • Include pulse-chase experiments with temporal control to distinguish between direct differentiation and cell division-coupled differentiation

• Functional validation approach:

  • Isolation of PROCR+ cells using FACS based on antibody detection or reporter systems

  • Single-cell and clonal expansion assays to assess multi-lineage differentiation capacity

  • Co-culture experiments with supporting cell types (e.g., endothelial cells) that might influence differentiation potential

• Experimental controls:

  • Include parallel tracing of established progenitor populations (e.g., PDX1+/NKX6.1+ cells)

  • Implement negative controls with cells known not to have progenitor capacity

  • Use randomized block design to account for variations between cell lines or donor tissue

• Temporal assessment:

  • Conduct time-course experiments spanning key developmental stages

  • Implement between-subjects design for different time points and within-subjects design for multi-parameter analysis

A comprehensive experimental design should include both in vitro differentiation assays and in vivo transplantation studies, with quantitative metrics for differentiation efficiency and functional maturation of resulting cell types.

What are the key challenges in reproducing findings related to PROCR+ progenitor cells, and how can researchers address them?

The reproducibility challenges surrounding PROCR+ progenitor cells stem from several factors:

Additionally, researchers should address the "groundbreaking report [that] did not have the expected impact in the field, as there were no follow-up reports from independent research groups recapitulating these findings" by:

• Systematically identifying potential variables that differed between the original study and subsequent attempts

• Sharing detailed protocols, reagents, and analysis pipelines

• Considering preregistration of replication studies to minimize publication bias

• Implementing multicenter collaborative studies with standardized protocols

Transparent reporting of both positive and negative results is essential to resolve these reproducibility challenges.

How does epigenetic regulation influence PROCR expression in human developmental contexts?

Understanding the epigenetic regulation of PROCR expression requires integrative analysis of chromatin accessibility, histone modifications, and DNA methylation patterns:

• Chromatin accessibility landscape:

  • Single-cell ATAC-seq analysis of human embryonic pancreas reveals distinct chromatin accessibility patterns in PROCR-expressing mesothelial cells

  • Key regulatory regions can be identified through motif discovery using tools like HOMER

• Transcription factor binding:

  • Analysis of accessible chromatin regions in PROCR+ cells reveals enrichment for binding motifs of specific transcription factor families

  • Comparison between mouse and human regulatory landscapes can identify conserved and species-specific regulation

• Developmental dynamics:

  • The PROCR-like transcriptional signature is gradually established during development, suggesting progressive epigenetic programming

  • Temporal analysis of chromatin states can identify pioneer factors that initially establish accessibility at the PROCR locus

• Experimental approaches:

  • CUT&RUN or CUT&Tag assays for specific histone modifications associated with active enhancers (H3K27ac) or repressed regions (H3K27me3)

  • CRISPR-based epigenetic editing to functionally validate regulatory elements

  • DNA methylation analysis of PROCR regulatory regions during differentiation

Understanding the epigenetic regulation may help explain why PROCR+ progenitor cells appear to be more prevalent in embryonic contexts compared to adult tissues, and could provide insights into methods for experimentally manipulating their differentiation potential.

What computational approaches are most effective for analyzing single-cell data to identify and characterize PROCR+ populations in human tissues?

Effective computational approaches for single-cell analysis of PROCR+ populations include:

• Quality control and preprocessing:

  • Filter low-quality cells (high mitochondrial content, low gene count)

  • Apply doublet removal algorithms (e.g., scDblFinder)

  • Implement normalization strategies appropriate for potentially rare cell populations

• Cell type identification and annotation:

  • Unsupervised clustering using appropriate resolution parameters

  • Integration of RNA-seq with ATAC-seq data when available

  • Transfer of cell type annotations between datasets using reference mapping

• Trajectory inference and developmental relationships:

  • Pseudotime ordering to reconstruct developmental progressions involving PROCR+ cells

  • RNA velocity analysis to infer differentiation directionality

  • Regulatory network reconstruction to identify key drivers of cell state transitions

• Multi-omics integration strategies:

  • Joint embedding of transcriptomic and epigenomic data

  • Correlation of gene expression with chromatin accessibility at promoters and enhancers

  • Integration of mouse and human datasets for evolutionary comparisons

• Visualization and interpretation:

  • UMAP or t-SNE for dimension reduction and visualization

  • Feature plots highlighting PROCR and co-expressed markers

  • Dot plots for comparing expression patterns across clusters

Researchers should also consider:

• Benchmarking multiple computational pipelines with simulated data

• Implementing computational controls to address batch effects

• Validating computational findings with orthogonal experimental approaches

What is the relationship between PROCR expression and cellular plasticity in human pancreatic development and disease?

The relationship between PROCR expression and cellular plasticity represents a frontier in understanding pancreatic development and potential regenerative capacity:

• Developmental plasticity:

  • PROCR+ cells in the embryonic pancreas demonstrate a unique transcriptional profile sharing both epithelial and mesenchymal characteristics

  • This dual identity may facilitate cellular transitions during organ development

  • The mesothelial identity of embryonic PROCR-like cells suggests potential involvement in epithelial-mesenchymal interactions during morphogenesis

• Disease contexts:

  • The relationship between embryonic PROCR+ mesothelial cells and the previously reported adult PROCR+ progenitors remains unclear

  • PROCR expression may be reactivated during tissue injury or stress conditions

  • Potential role in pancreatic remodeling during diabetes progression requires further investigation

• Experimental assessment of plasticity:

  • Lineage tracing combined with injury models to assess fate potential under stress conditions

  • Single-cell multi-omic profiling before and after experimental perturbations

  • Comparative analysis with other populations showing differentiation plasticity

• Therapeutic implications:

  • Understanding the mechanisms underlying PROCR-associated plasticity could inform regenerative medicine approaches

  • The spontaneous differentiation of PROCR-like cells during in vitro directed differentiation of human pluripotent stem cells suggests potential for manipulation in therapeutic contexts

The apparent discrepancy between initial reports of adult PROCR+ progenitors and subsequent studies failing to reproduce these findings highlights the complexity of cellular plasticity and the need for rigorous experimental approaches to delineate the true nature and potential of PROCR-expressing cells in human pancreatic biology.

What emerging technologies will advance our understanding of PROCR function in human tissues?

Several cutting-edge technologies show promise for deepening our understanding of PROCR biology:

• Spatial transcriptomics and proteomics: Technologies like Visium, MERFISH, or Co-Detection by Indexing (CODEX) will provide spatial context to PROCR expression, revealing microenvironmental influences and tissue-specific interactions not captured by dissociated single-cell analyses.

• Multi-modal single-cell profiling: Simultaneous measurement of transcriptome, proteome, and epigenome from the same cells will provide integrated views of PROCR regulation and function.

• Organoid-on-a-chip platforms: Microfluidic systems combining multiple tissue types will enable studies of PROCR+ cell interactions with endothelial, immune, or stromal components under dynamic conditions.

• In vivo human models: Advanced humanized mouse models and chimeric approaches may provide deeper insights into PROCR function in physiologically relevant contexts.

• AI-enhanced image analysis: Machine learning approaches for analyzing complex tissue architecture and cell-cell interactions will help characterize PROCR+ cells within their native niches.

Implementation of these technologies will require carefully designed experimental protocols with appropriate controls and validation strategies to ensure reproducible and meaningful results .

How might understanding PROCR+ cell populations contribute to regenerative medicine approaches for diabetes?

The potential applications of PROCR research to regenerative medicine for diabetes include:

• Optimized differentiation protocols: Insights from PROCR-expressing mesothelial cells during pancreatic development could inform refinements to current protocols for generating insulin-producing cells from pluripotent stem cells, which "still requires optimization" .

• Identification of supporting cell types: The embryonic mesothelial origin of PROCR-like cells suggests potential non-epithelial contributions to pancreatic development that might be leveraged in vitro.

• In vivo regeneration strategies: If specific signals governing PROCR+ cell function can be identified, they might be applied to stimulate endogenous regenerative processes in diabetic patients.

• Biomarkers for differentiation efficiency: PROCR expression patterns could serve as indicators of successful developmental progression during in vitro differentiation protocols.

• Disease modeling platforms: PROCR-expressing populations might provide novel insights into developmental origins of pancreatic dysfunction.