Study of Somite Formation Model Based on Microfluidic System

Background and Research Motivation

The formation of somites plays a crucial role in vertebrate embryonic development, significantly influencing the layout and functionality of the embryonic musculoskeletal system. Somite formation involves the segmentation of bilateral presomitic mesoderm (PSM) into symmetrical epithelial somites in a head-to-tail direction. During this process, the gradient changes in biochemical signals (such as fibroblast growth factor FGF and retinoic acid RA) and biomechanical influences are essential. However, most existing somite formation models use suspension cultures, lacking precise control over biochemical gradients and mechanical signals, limiting the study of complex biochemical-biomechanical interactions.

Against this backdrop, a research team from the University of Michigan and Harvard University collaborated to construct a presomitic mesoderm (PSM) model from human pluripotent stem cells (hPSCs) using a microfluidic device. This model introduced exogenous biochemical gradients, achieving spatial pattern control of somite formation. The team systematically investigated the regulatory role of biomechanics in somite formation, proposing a scaling law based on mechanical models for controlling somite size and revealing the roles of cell adhesion, force generation, and epithelial-mesenchymal transition as mechanical regulatory factors in somite formation. These findings were published in the journal “Cell Stem Cell,” marking significant progress in somite formation research.

Research Process and Methods

1. Construction of the Somite Model and Design of the Microfluidic Device

The research team designed a PDMS (polydimethylsiloxane)-based microfluidic device comprising three channels. The bottom of the middle channel featured multiple microgrooves used to position and confine hPSC-derived PSM tissue, simulating the mechanical boundary conditions of somite formation. By adding signaling molecules such as FGF, RA, and Wnt to different reservoirs, stable biochemical gradients were formed in the PSM tissue through passive diffusion. Fluorescent labeling diffusion experiments confirmed the stability of the gradient formation over approximately 36 hours.

2. Real-Time Imaging of Somite Formation and Marker Detection

Using fluorescence microscopy, researchers tracked the process of somite formation and observed somite segmentation from head to tail in the model. Immunofluorescence staining further validated the expression patterns of markers like Pax3 and Tbx6 during somite formation. Tbx6 was primarily expressed in the PSM region, while Pax3 was expressed in the formed somites, with the spatial expression pattern gradually extending toward the tail over time.

3. Biomechanical Regulation through a Mechanical Model

During the research, the team constructed a mechanical model based on the biomechanical processes involved in somite formation. The model hypothesizes that in the head region of the PSM, as cells undergo epithelialization and transition into somites, the somite precursor gradually contracts, creating a tissue boundary with stored strain energy. When the strain energy exceeds the surface energy needed for somite formation, separation occurs between the PSM and the new somite, forming a new somite. Through this model, researchers proposed a scaling law revealing the relationship between somite size and PSM length.

4. Single-Cell Transcriptomics Analysis

To explore the dynamics of gene expression during somite formation, the team conducted single-cell RNA sequencing on cells in the model at different time points. The results showed that specific genes exhibited gradual regulatory patterns as PSM cells transitioned into somite cells. The expression of genes like Tbx6, Foxc2, and Pax3 changed progressively with the transformation of cell fate during somite formation.

5. Testing Mechanical Influences in Somite Formation

The team also validated the role of mechanical regulation in somite formation through mechanical, chemical, and genetic perturbation experiments. For example, applying periodic tensile strain via a microfluidic device revealed that somite size decreased with increased strain. Inhibiting cytoskeletal contraction or cell adhesion significantly decreased somite formation efficiency, further demonstrating the importance of mechanical factors in somite formation.

Research Results

Biomechanical Regulation of Somite Formation

The findings indicate that somite formation is a complex process jointly regulated by biochemical and mechanical factors. By constructing a somite formation model and introducing exogenous biochemical gradients and mechanical constraints, researchers were able to simulate key steps in somite formation in a vitro system. The scaling law based on strain energy and surface energy provided a satisfactory explanation for somite size regulation mechanisms, supported by somite formation data from animal models like mice, chickens, and zebrafish.

Dynamic Transcriptomics via Single-Cell RNA Sequencing

The single-cell RNA sequencing results revealed significant changes in specific gene expression as PSM cells transitioned into somite cells, reflecting a tight association between the biochemical gradient and cell fate differentiation during somite formation. This provides new perspectives for understanding the molecular mechanisms in somite formation.

Mechanical and Chemical Perturbations in Somite Formation

Mechanical interventions showed that the somite formation region could be regulated by stress changes, indicating the role of mechanical signals in somite size and segmentation. Furthermore, chemical inhibition of cytoskeletal contraction significantly inhibited somite formation, highlighting the importance of cellular contractile and adhesive forces in somite formation. Genetic knockout experiments further affirmed the pivotal role of certain genes (like Tcf15) in somite epithelialization and boundary formation regulation.

Research Significance

This study successfully constructed a somite formation model in a microfluidic system, simulating key steps in human somite formation in vitro, providing a novel tool and research approach to understand the biochemical-biomechanical interactions in vertebrate somite formation. Compared to existing somite models, the proposed model allows for more effective control of exogenous signals and mechanical boundaries, enabling the separation and in-depth exploration of various factors affecting somite formation.

The study not only unveils scaling laws and biomechanical regulation mechanisms in somite formation but also offers critical experimental methodologies for further somite research. Especially in research on human musculoskeletal developmental disorders and related diseases, this model has potential application value. In the future, incorporating more dynamic signals and four-dimensional imaging technologies is expected to further uncover the molecular and cellular level details of the somite formation process, providing richer data support for understanding the fundamental mechanisms of human development.

Conclusion

This study successfully developed a precisely controlled somite formation model using a microfluidic system, simulating the biomechanical processes of human somite formation in vitro. The findings support the significance of biochemical-mechanical interactions in somite formation and propose a new scaling law to interpret the regulatory mechanism of somite size. This research holds significant guiding significance for future developmental biology research and provides a new experimental basis for the study of human musculoskeletal system development and diseases.