Identifying distinct nanoscopic features of native collagen fibrils towards early diagnosis of pelvic organ prolapse.

Pelvic organ prolapse (POP) is characterized by weakening of the connective tissues and loss of support for the pelvic organs. Collagen is the predominant, load-bearing protein within pelvic floor connective tissues. In this study, we examined the nanoscopic structures and biomechanics of native collagen fibrils in surgical, vaginal wall connective tissues from healthy women and POP patients. Compared to controls, collagen fibrils in POP samples were bulkier, more uneven in width and stiffer with aberrant D-period. Additionally, the ratio of collagen I (COLI) and collagen III (COLIII) is doubled in POP with a concomitant reduction of the amount of total collagen. Thus, POP is characterized by abnormal biochemical composition and biophysical characteristics of collagen fibrils that form a loose and fragile fiber network accountable for the weak load-bearing capability. The study identifies nanoscale alterations in collagen as diagnostic markers that could enable pre-symptomatic or early diagnosis of POP. FROM THE CLINICAL EDITOR: Pelvic organ prolapse (POP) occurs due to abnormalities of the supporting connective tissues. The underlying alterations of collagen fibers in the connective tissues have not been studied extensively. In this article, the authors showed that collagen fibrils in POP patients were much different from normal controls. The findings may provide a framework for the diagnosis of other connective diseases.

Matrix-specified differentiation of human decidua parietalis placental stem cells.

To create suitable biological scaffolds for tissue engineering and cell therapeutics, it is essential to understand the matrix-mediated specification of stem cell differentiation. To this end, we studied the effect of collagen type I on stem cell lineage specification. We altered the properties of collagen type I by incorporating carbon nanotubes (CNT). The collagen-CNT composite material was stiffer with thicker fibers and longer D-period. Human decidua parietalis stem cells (hdpPSC) were found to differentiate exclusively and rapidly towards neural cells on the collagen-CNT matrix. We attribute this accelerated neural differentiation to the enhanced structural and mechanical properties of collagen-CNT material. Strikingly, the collagen-CNT matrix, unlike collagen, imposes the neural fate by an alternate mechanism that may be independent of beta-1 integrin and beta-catenin. The study demonstrates the sensitivity of stem cells to subtle changes in the matrix and the utilization of a novel biocomposite material for efficient and directed differentiation of stem cells.

Analysis of affinity maps of membrane proteins on individual human embryonic stem cells.

The heterogeneity found in many cell types has greatly inspired research in single-cell gene and protein profiling for discovering the origin of heterogeneity and its role in cell fate decisions. Among the existing techniques to probe heterogeneity, atomic force microscopy (AFM) utilizes an antibody/ligand-modified tip to explore the distribution of a target membrane protein on individual cells in their native environment. In this paper, we establish a practical model to analyze the data systematically, and attempt the quantification of membrane protein abundance on single cells by taking account issues, such as the level of nonspecific interaction, the probe resolution, and the reproducibility of detecting protein distribution. We demonstrated the application in examining the heterogeneous distribution and the local protein abundance of TRA-1-81 antigen on human embryonic stem (hES) cells at the subcellular level. Heterogeneity in TRA-1-81 expression was also detected at the single cell level, suggesting the presence of subpopulation cells within an undifferentiated hES cell colony. The method provides a platform to unveiling the correlation between heterogeneity of membrane proteins and cell development in a complex cell community.

Adapting collagen/CNT matrix in directing hESC differentiation.

The lineage selection in human embryonic stem cell (hESC) differentiation relies on both the growth factors and small molecules in the media and the physical characteristics of the micro-environment. In this work, we utilized various materials, including the collagen-carbon nanotube (collagen/CNT) composite material, as cell culture matrices to examine the impact of matrix properties on hESC differentiation. Our AFM analysis indicated that the collagen/CNT formed rigid fibril bundles, which polarized the growth and differentiation of hESCs, resulting in more than 90% of the cells to the ectodermal lineage in Day 3 in the media commonly used for spontaneous differentiation. We also observed the differentiated cells followed the coarse alignment of the collagen/CNT matrix. The research not only revealed the responsiveness of hESCs to matrix properties, but also provided a simple yet efficient way to direct the hESC differentiation, and imposed the potential of forming neural-cell based bio-devices for further applications.

Effect of CNT on collagen fiber structure, stiffness assembly kinetics and stem cell differentiation.

Collagen is a native one-dimensional nanomaterial. Carbon nanotube (CNT) was found to interface with biological materials and show promising applications in creating reinforced scaffolds for tissue engineering and regenerative medicine. In this study, we examined the unique role of CNT in collagen fiber structure, mechanical strength and assembly kinetics. The results imply that CNT interacts with collagen at the molecular level. It relaxes the helical coil of collagen fibrils and has the effect of flattening the fibers leading to the elongation of D-period, the characteristic banding feature of collagen fibers. The surface charge of oxidized CNT leads to enhanced local ionic strength during collagen fibrillogenesis, accounting for the slower kinetics of collagen-CNT (COL-CNT) fiber assembly and the formation of thicker fibers. Due to the rigidity of CNT, the addition of CNT increases the fiber stiffness significantly. When applied as a matrix for human decidua parietalis placental stem cells (hdpPSCs) differentiation, COL-CNT was found to support fast and efficient neural differentiation ascribed to the elongated D-period. These results highlight the superiority of CNT to modulate collagen fiber assembly at the molecular level. The study also exemplifies the use of CNT to enhance the functionality of collagen for biological and biomedical applications.

Structural and mechanical profiles of native collagen fibers in vaginal wall connective tissues.

Collagen, an ubiquitous biomaterial, confers robustness and resilience to connective tissues. In this study, we analyzed the structure and elasticity profile of collagen from the vaginal wall connective tissue of healthy pre-menopausal (pre-M) and postmenopausal (post-M) women. The histological staining assisted study with an atomic force microscope renders the examination of native collagen fibers on site of the connective tissue from nanoscopic scale to microscopic scale with high spatial resolution. Our results suggest that during menopause, collagen's structure and elasticity are subject to changes at all levels of organization- between individual collagen fibers, between collagen and muscle, and between collagen and other matrix elements. The systematic analysis of the native structure and mechanical properties of collagen within a tissue provides a potential way to study non-fatal conditions such as pelvic organ prolapse and other genito-urinary disorders, where the initial symptoms are subtle and multivariate, and where early detection of patient's condition may allow better non-invasive interventions and reduce the number of women undergoing surgical correction of these common disorders.

Spatially resolved quantification of E-cadherin on target hES cells.

The local expression and distribution pattern of protein on a cell play essential roles in signal transduction within a cell or between cells. Here we report on the development of a spatially resolved quantification method, which was applied in the study of E-cadherin local expression in identified undifferentiated and differentiated human embryonic stem (hES) cells in their native cellular environment. This was achieved by a novel immunofluorescence assisted affinity mapping (IF-AM) method, in which immunofluorescence provides the guidance to locate a desired type of cell in a cell community for performing affinity mapping to quantify the local protein density. The results unveiled the crucial role of E-cadherin in mediating hES cell proliferation and differentiation: the expression of E-cadherin is markedly higher on undifferentiated cells, and the growth of hES cells in unique colonies is contingent on the homogeneous distribution of E-cadherin. Due to the ability of directly assessing individual proteins of a cell, the IF-AM method is shown to be a sensitive tool for resolving subtle differences in the local expression of membrane proteins even at low abundance.