comparative-study-basement-membrane-matrices-for-human-stem-cell

Comparative Study of Basement-Membrane Matrices for Human Stem Cell Maintenance and Intestinal Organoid Generation

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) ...

University of Florida

The organization of proteins is an important determinant of functionality in soft tissues. However, such organization is difficult to monitor over time in soft tissue with complex compositions. Here, we establish a method to determine the alignment of proteins in soft tissues of varying composition by polarized Raman spectroscopy (PRS). Unlike most conventional microscopy methods, PRS leverages non-destructive, label-free sample preparation. PRS data from highly aligned muscle layers were utilized to derive a weighting function for aligned proteins via principal component analysis (PCA). This trained weighting function was used as a master loading function to calculate a principal component score (PC1 Score) as a function of polarized angle for tendon, dermis, hypodermis, and fabricated collagen gels. Since the PC1 Score calculated at arbitrary angles was insufficient to determine level of alignment, we developed an Amplitude Alignment Metric by fitting a sine function to PC1 Score with respect to polarized angle. We found that our PRS-based Amplitude Alignment Metric can be used as an indicator of level of protein alignment in soft tissues in a non-destructive manner with label-free preparation and has similar discriminatory capacity among isotropic and anisotropic samples compared to microscopy-based image processing method. This PRS method does not require a priori knowledge of sample orientation nor composition and appears insensitive to changes in protein composition among different tissues. The Amplitude Alignment Metric introduced here could enable convenient and adaptable evaluation of protein alignment in soft tissues of varying protein and cell composition. STATEMENT OF SIGNIFICANCE: Polarized Raman spectroscopy (PRS) has been used to characterize the of organization of soft tissues. However, most of the reported applications of PRS have been on collagen-rich tissues and reliant on intensities of collagen-related vibrations. This work describes a PRS method via a multivariate analysis to characterize alignment in soft tissues composed of varying proteins. Of note, the highly aligned muscle layer of mouse skin was used to train a master function then applied to other soft tissue samples, and the degree of anisotropy in the PRS response was evaluated to obtain the level of alignment in tissues. We have demonstrated that this method supports convenient and adaptable evaluation of protein alignment in soft tissues of varying protein and cell composition.

Label-free quantification of soft tissue alignment by polarized Raman spectroscopy.

Altered extracellular matrix (ECM) production is a hallmark of many fibroproliferative diseases, including certain cancers. The high incidence of glycan-rich components within altered ECM makes the use of glycan-binding proteins such as Galectin-3 (G3) a promising therapeutic strategy. The complexity of ECM as a rich 3D network of proteins with varied glycosylation states makes it challenging to determine the retention of glycan-binding proteins in altered ECM environments. Computational models capable of predicting the transport of glycan-binding proteins in altered ECM can benefit the design and testing of such proteins and associated novel therapeutic strategies. However, such computational models require many kinetic parameters that cannot be estimated from traditional 2D pharmacokinetic assays. To validate transport properties of G3 in 3D ECM constructs, we developed a species transport model that includes diffusion and matrix-binding components to predict retention of G3 fusion proteins in glycan-rich ECM. By iteratively comparing our computational model to experimental results, we are able to determine a reasonable range of parameters for a robust computational model of G3 transport. We anticipate this overall approach to building a data-driven model is translatable to other ECM-targeting therapeutic strategies.

Experimental and Computational Models of Transport of Galectin-3 Through Glycosylated Matrix.

The extracellular matrix (ECM) provides both physical and chemical cues that dictate cell function and contribute to muscle maintenance. Muscle cells require efficient mitochondria to satisfy their high energy demand, however, the role the ECM plays in moderating mitochondrial function is not clear. We hypothesized that the ECM produced by stromal cells with mitochondrial dysfunction (Barth syndrome, BTHS) provides cues that contribute to metabolic dysfunction independent of muscle cell health. To test this, we harnessed the ECM production capabilities of human pluripotent stem-cell-derived cardiac fibroblasts (hPSC-CFs) from healthy and BTHS patients to fabricate cell-derived matrices (CDMs) with controlled topography, though we found that matrix composition from healthy versus diseased cells influenced myotube formation independent of alignment cues. To further investigate the effects of matrix composition, we then examined the influence of healthy- and BTHS-derived CDMs on myotube formation and metabolic function. We found that BTHS CDMs induced lower fusion index, lower ATP production, lower mitochondrial membrane potential, and higher ROS generation than the healthy CDMs. These findings imply that BTHS-derived ECM alone contributes to myocyte dysfunction in otherwise healthy cells. Finally, to investigate potential mechanisms, we defined the composition of CDMs produced by hPSC-CFs from healthy and BTHS patients using mass spectrometry and identified 15 ECM and related proteins that were differentially expressed in the BTHS-CDM compared to healthy CDM. Our results highlight that ECM composition affects skeletal muscle formation and metabolic efficiency in otherwise healthy cells, and our methods to generate patient-specific CDMs are a useful tool to investigate the influence of the ECM on disease progression and to investigate variability among diseased patients. STATEMENT OF SIGNIFICANCE: Muscle function requires both efficient metabolism to generate force and structured extracellular matrix (ECM) to transmit force, and we sought to examine the interactions between metabolism and ECM when metabolic disease is present. We fabricated patient-specific cell derived matrices (CDMs) with controlled topographic features to replicate the composition of healthy and mitochondrial-diseased (Barth syndrome) ECM. We found that disease-derived ECM negatively affects metabolic function of otherwise healthy myoblasts, and we identified several proteins in disease-derived ECM that may be mediating this dysfunction. We anticipate that our patient-specific CDM system could be fabricated with other topographies and cell types to study cell functions and diseases of interest beyond mitochondrial dysfunction and, eventually, be applied toward personalized medicine.

Matrix produced by diseased cardiac fibroblasts affects early myotube formation and function.

Monitoring of extracellular matrix (ECM) microstructure is essential in studying structure-associated cellular processes, improving cellular function, and for ensuring sufficient mechanical integrity in engineered tissues. This paper describes a novel method to study the microscale alignment of the matrix in engineered tissue scaffolds (ETS) that are usually composed of a variety of biomacromolecules derived by cells. First, a trained loading function was derived from Raman spectra of highly aligned native tissue via principal component analysis (PCA), where prominent changes associated with specific Raman bands (e.g., 1444, 1465, 1605, 1627-1660, and 1665-1689 cm) were detected with respect to the polarization angle. These changes were mainly caused by the aligned matrix of many compounds within the tissue relative to the laser polarization, including proteins, lipids, and carbohydrates. Hence this trained function was applied to quantify the alignment within ETS of various matrix components derived by cells. Furthermore, a simple metric called Amplitude Alignment Metric (AAM) was derived to correlate the orientation dependence of polarized Raman spectra of ETS to the degree of matrix alignment. It was found that the AAM was significantly higher in anisotropic ETS than isotropic ones. The PRS method revealed a lower -value for distinguishing the alignment between these two types of ETS as compared to the microscopic method for detecting fluorescent-labeled protein matrices at a similar microscopic scale. These results indicate that the anisotropy of a complex matrix in engineered tissue can be assessed at the microscopic scale using a PRS-based simple metric, which is superior to the traditional microscopic method. This PRS-based method can serve as a complementary tool for the design and assessment of engineered tissues that mimic the native matrix organizational microstructures.

Article	Year
Label-free quantification of soft tissue alignment by polarized Raman spectroscopy. Acta biomaterialia\| PubMed ID: 34537413	2021
Experimental and Computational Models of Transport of Galectin-3 Through Glycosylated Matrix. Annals of biomedical engineering\| PubMed ID: 35352215	2022
Matrix produced by diseased cardiac fibroblasts affects early myotube formation and function. Acta biomaterialia\| PubMed ID: 36055608	2022
Label-Free Quantification of Microscopic Alignment in Engineered Tissue Scaffolds by Polarized Raman Spectroscopy. ACS biomaterials science & engineering\| PubMed ID: 37170804	2023
Editorial: Insights in drug metabolism and transport: 2021. Frontiers in pharmacology\| PubMed ID: 37229271	2023

Janny Pineiro-Llanes

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