Developmental morphogens direct human induced pluripotent stem cells toward an annulus fibrosus-like cell phenotype.

Introduction: Therapeutic interventions for intervertebral disc herniation remain scarce due to the inability of endogenous annulus fibrosus (AF) cells to respond to injury and drive tissue regeneration. Unlike other orthopedic tissues, such as cartilage, delivery of exogenous cells to the site of annular injury remains underdeveloped, largely due to a lack of an ideal cell source and the invasive nature of cell isolation. Human induced pluripotent stem cells (iPSCs) can be differentiated to specific cell fates using biochemical factors and are, therefore, an invaluable tool for cell therapy approaches. While differentiation protocols have been developed for cartilage and fibrous connective tissues (e.g., tendon), the signals that regulate the induction and differentiation of human iPSCs toward the AF fate remain unknown. Methods: iPSC-derived sclerotome cells were treated with various combinations of developmental signals including transforming growth factor beta 3 (TGF-ß3), connective tissue growth factor (CTGF), platelet derived growth factor BB (PDGF-BB), insulin-like growth factor 1 (IGF-1), or the Hedgehog pathway activator, Purmorphamine, and gene expression changes in major AF-associated ECM genes were assessed. The top performing combination treatments were further validated by using three distinct iPSC lines and by assessing the production of upregulated ECM proteins of interest. To conduct a broader analysis of the transcriptomic shifts elicited by each factor combination, and to compare genetic profiles of treated cells to mature human AF cells, a 96.96 Fluidigm gene expression array was applied, and principal component analysis was employed to identify the transcriptional signatures of each cell population and treatment group in comparison to native AF cells. Results: TGF-ß3, in combination with PDGF-BB, CTGF, or IGF-1, induced an upregulation of key AF ECM genes in iPSC-derived sclerotome cells. In particular, treatment with a combination of TGF-ß3 with PDGF-BB for 14 days significantly increased gene expression of collagen II and aggrecan and increased protein deposition of collagen I and elastin compared to other treatment groups. Assessment of genes uniquely highly expressed by AF cells or SCL cells, respectively, revealed a shift toward the genetic profile of AF cells with the addition of TGF-ß3 and PDGF-BB for 14 days. Discussion: These findings represent an initial approach to guide human induced pluripotent stem cells toward an AF-like fate for cellular delivery strategies.

Reduction in postnatal weight-bearing does not alter the trajectory of murine meniscus growth and maturation.

The early postnatal period represents a critical window for the maturation and development of orthopedic tissues, including those within the knee joint. To understand how mechanical loading impacts the maturational trajectory of the meniscus and other tissues of the hindlimb, perturbation of postnatal weight bearing was achieved through surgical resection of the sciatic nerve in neonatal mice at 1 or 14 days old. Sciatic nerve resection (SNR) produced significant and persistent disruptions in gait, leading to reduced tibial length and reductions in Achilles tendon mechanical properties. However, SNR resulted in minimal disruptions in morphometric parameters of the menisci and other structures in the knee joint, with no detectable differences in Col1a1-YFP or Col2a1-CFP expressing cells within the menisci. Furthermore, micromechanical properties of the meniscus and cartilage (as assessed by atomic force microscopy-based nanoindentation testing) were not different between experimental groups. In contrast to our initial hypothesis, reduced hindlimb weight bearing via neonatal SNR did not significantly impact the growth and development of the knee meniscus. This unexpected finding demonstrates that the input mechanical threshold required to sustain meniscus development may be lower than previously hypothesized, though future studies incorporating skeletal kinematic models coupled with force plate measurements will be required to calculate the loads passing through the affected hindlimb and precisely define these thresholds. Collectively, these results provide insight into the mechanobiological responses of the meniscus to alterations in load, and contribute to our understanding of the factors that influence normal postnatal development.

Lack of skeletal muscle contraction disrupts fibrous tissue morphogenesis in the developing murine knee.

Externally applied forces, such as those generated through skeletal muscle contraction, are important to embryonic joint formation, and their loss can result in gross morphologic defects including joint fusion. While the absence of muscle contraction in the developing chick embryo leads to dissociation of dense connective tissue structures of the knee and ultimately joint fusion, the central knee joint cavitates whereas the patellofemoral joint does not in murine models lacking skeletal muscle contraction, suggesting a milder phenotype. These differential results suggest that muscle contraction may not have as prominent of a role in the growth and development of dense connective tissues of the knee. To explore this question, we investigated the formation of the menisci, tendon, and ligaments of the developing knee in two murine models that lack muscle contraction. We found that while the knee joint does cavitate, there were multiple abnormalities in the menisci, patellar tendon, and cruciate ligaments. The initial cellular condensation of the menisci was disrupted and dissociation was observed at later embryonic stages. The initial cell condensation of the tendon and ligaments were less affected than the meniscus, but these tissues contained cells with hyper-elongated nuclei and displayed diminished growth. Interestingly, lack of muscle contraction led to the formation of an ectopic ligamentous structure in the anterior region of the joint as well. These results indicate that muscle forces are essential for the continued growth and maturation of these structures during this embryonic period.

Intrinsic and growth-mediated cell and matrix specialization during murine meniscus tissue assembly.

The incredible mechanical strength and durability of mature fibrous tissues and their extremely limited turnover and regenerative capacity underscores the importance of proper matrix assembly during early postnatal growth. In tissues with composite extracellular matrix (ECM) structures, such as the adult knee meniscus, fibrous (Collagen-I rich), and cartilaginous (Collagen-II, proteoglycan-rich) matrix components are regionally segregated to the outer and inner portions of the tissue, respectively. While this spatial variation in composition is appreciated to be functionally important for resisting complex mechanical loads associated with gait, the establishment of these specialized zones is poorly understood. To address this issue, the following study tracked the growth of the murine meniscus from its embryonic formation through its first month of growth, encompassing the critical time-window during which animals begin to ambulate and weight bear. Using histological analysis, region specific high-throughput qPCR, and Col-1, and Col-2 fluorescent reporter mice, we found that matrix and cellular features defining specific tissue zones were already present at birth, before continuous weight-bearing had occurred. These differences in meniscus zones were further refined with postnatal growth and maturation, resulting in specialization of mature tissue regions. Taken together, this work establishes a detailed timeline of the concurrent spatiotemporal changes that occur at both the cellular and matrix level throughout meniscus maturation. The findings of this study provide a framework for investigating the reciprocal feedback between cells and their evolving microenvironments during assembly of a mechanically robust fibrocartilage tissue, thus providing insight into mechanisms of tissue degeneration and effective regenerative strategies.

Nuclear envelope wrinkling predicts mesenchymal progenitor cell mechano-response in 2D and 3D microenvironments.

Exogenous mechanical cues are transmitted from the extracellular matrix to the nuclear envelope (NE), where mechanical stress on the NE mediates shuttling of transcription factors and other signaling cascades that dictate downstream cellular behavior and fate decisions. To systematically study how nuclear morphology can change across various physiologic microenvironmental contexts, we cultured mesenchymal progenitor cells (MSCs) in engineered 2D and 3D hyaluronic acid hydrogel systems. Across multiple contexts we observed highly 'wrinkled' nuclear envelopes, and subsequently developed a quantitative single-cell imaging metric to better evaluate how wrinkles in the nuclear envelope relate to progenitor cell mechanotransduction. We determined that in soft 2D environments the NE is predominately wrinkled, and that increases in cellular mechanosensing (indicated by cellular spreading, adhesion complex growth, and nuclear localization of YAP/TAZ) occurred only in absence of nuclear envelope wrinkling. Conversely, in 3D hydrogel and tissue contexts, we found NE wrinkling occurred along with increased YAP/TAZ nuclear localization. We further determined that these NE wrinkles in 3D were largely generated by actin impingement, and compared to other nuclear morphometrics, the degree of nuclear wrinkling showed the greatest correlation with nuclear YAP/TAZ localization. These findings suggest that the degree of nuclear envelope wrinkling can predict mechanotransduction state in mesenchymal progenitor cells and highlights the differential mechanisms of NE stress generation operative in 2D and 3D microenvironmental contexts.

Aberrant mechanosensing in injured intervertebral discs as a result of boundary-constraint disruption and residual-strain loss.

In fibrous tissues, prestressed boundary constraints at bone interfaces instil residual strain throughout the tissue, even when unloaded. For example, internal swelling pressures in the central nucleus pulposus of the intervertebral disc generate prestrain in the outer annulus fibrosus. With injury and depressurization, these residual strains are lost. Here we show that the loss of residual strains in the intervertebral disc alters the microenvironment and instigates aberrant tissue remodelling and the adoption of atypical cellular phenotypes. By using puncture surgery of the annulus fibrosus in rabbits, ex vivo puncture experiments and electrospun nanofibrous scaffolds recapitulating these evolving boundary constraints, we show that the loss of residual strain promotes short-term apoptosis and the emergence of a fibrotic phenotype. We also show that local fibre organization and cellular contractility mediate this process and that the aberrant cellular changes could be abrogated by targeting the cell-mechanosensing machinery with small molecules. Our findings indicate that injury to dense connective tissues under prestrain alters boundary constraints and residual strain; this leads to aberrant mechanosensing, which in turn promotes disease progression.

The CENP-L-N Complex Forms a Critical Node in an Integrated Meshwork of Interactions at the Centromere-Kinetochore Interface.

During mitosis, the macromolecular kinetochore complex assembles on the centromere to orchestrate chromosome segregation. The properties and architecture of the 16-subunit Constitutive Centromere-Associated Network (CCAN) that allow it to build a robust platform for kinetochore assembly are poorly understood. Here, we use inducible CRISPR knockouts and biochemical reconstitutions to define the interactions between the human CCAN proteins. We find that the CCAN does not assemble as a linear hierarchy, and instead, each sub-complex requires multiple non-redundant interactions for its localization to centromeres and the structural integrity of the overall assembly. We demonstrate that the CENP-L-N complex plays a crucial role at the core of this assembly through interactions with CENP-C and CENP-H-I-K-M. Finally, we show that the CCAN is remodeled over the cell cycle such that sub-complexes depend on their interactions differentially. Thus, an interdependent meshwork within the CCAN underlies the centromere specificity and stability of the kinetochore.

Kinetochore genes are coordinately up-regulated in human tumors as part of a FoxM1-related cell division program.

The key player in directing proper chromosome segregation is the macromolecular kinetochore complex, which mediates DNA-microtubule interactions. Previous studies testing individual kinetochore genes documented examples of their overexpression in tumors relative to normal tissue, leading to proposals that up-regulation of specific kinetochore genes may promote tumor progression. However, kinetochore components do not function in isolation, and previous studies did not comprehensively compare the expression behavior of kinetochore components. Here we analyze the expression behavior of the full range of human kinetochore components in diverse published expression compendia, including normal tissues and tumor samples. Our results demonstrate that kinetochore genes are rarely overexpressed individually. Instead, we find that core kinetochore genes are coordinately regulated with other cell division genes under virtually all conditions. This expression pattern is strongly correlated with the expression of the forkhead transcription factor FoxM1, which binds to the majority of cell division promoters. These observations suggest that kinetochore gene up-regulation in cancer reflects a general activation of the cell division program and that altered expression of individual kinetochore genes is unlikely to play a causal role in tumorigenesis.

Comparison of mechanical properties of human ascending aorta and aortic sinuses.

BACKGROUND: Computational finite element models of the aortic root have previously used material properties of the ascending aorta to describe both aortic sinuses and ascending aorta. We have previously demonstrated significant material property differences between ascending aorta and sinuses in pigs. However, it is unknown whether these regional material property differences exist in humans. The main objective of this study was to investigate biomechanics of fresh human ascending aorta and aortic sinuses and compare nonlinear material properties of these regions. METHODS: Fresh human aortic root specimens obtained from the California Transplant Donor Network (Oakland, CA) were subjected to displacement-controlled equibiaxial stretch testing within 24 hours of harvest. Stress-strain data recorded were used to derive strain energy functions for each region. Tissue behavior was quantified by tissue stiffness and a direct comparison was made between different regions of aortic root at physiologic stress levels. RESULTS: All regions demonstrated a nonlinear response to strain during stretch testing in both circumferential and longitudinal directions. No significant difference in tissue stiffness was found between anterior and posterior regions of the ascending aorta or among the three sinuses in both directions. However, our results demonstrated that human ascending aorta is significantly more compliant than aortic sinuses in both circumferential and longitudinal directions within the physiologic stress range. CONCLUSIONS: Significant material and structural differences were observed between human ascending aorta and aortic sinuses. Regionally specific material properties should be employed in computational models used to assess treatments of structural aortic root disease.

Responsive micromolds for sequential patterning of hydrogel microstructures.

Microscale hydrogels have been shown to be beneficial for various applications such as tissue engineering and drug delivery. A key aspect in these applications is the spatial organization of biological entities or chemical compounds within hydrogel microstructures. For this purpose, sequentially patterned microgels can be used to spatially organize either living materials to mimic biological complexity or multiple chemicals to design functional microparticles for drug delivery. Photolithographic methods are the most common way to pattern microscale hydrogels but are limited to photocrosslinkable polymers. So far, conventional micromolding approaches use static molds to fabricate structures, limiting the resulting shapes that can be generated. Herein, we describe a dynamic micromolding technique to fabricate sequentially patterned hydrogel microstructures by exploiting the thermoresponsiveness of poly(N-isopropylacrylamide)-based micromolds. These responsive micromolds exhibited shape changes under temperature variations, facilitating the sequential molding of microgels at two different temperatures. We fabricated multicompartmental striped, cylindrical, and cubic microgels that encapsulated fluorescent polymer microspheres or different cell types. These responsive micromolds can be used to immobilize living materials or chemicals into sequentially patterned hydrogel microstructures which may potentially be useful for a range of applications at the interface of chemistry, materials science and engineering, and biology.

Responsive microgrooves for the formation of harvestable tissue constructs.

Given its biocompatibility, elasticity, and gas permeability, poly(dimethylsiloxane) (PDMS) is widely used to fabricate microgrooves and microfluidic devices for three-dimensional (3D) cell culture studies. However, conformal coating of complex PDMS devices prepared by standard microfabrication techniques with desired chemical functionality is challenging. This study describes the conformal coating of PDMS microgrooves with poly(N-isopropylacrylamide) (PNIPAAm) by using initiated chemical vapor deposition (iCVD). These microgrooves guided the formation of tissue constructs from NIH-3T3 fibroblasts that could be retrieved by the temperature-dependent swelling property and hydrophilicity change of the PNIPAAm. The thickness of swollen PNIPAAm films at 24 °C was approximately 3 times greater than at 37 °C. Furthermore, PNIPAAm-coated microgroove surfaces exhibit increased hydrophilicity at 24 °C (contact angle θ = 30° ± 2) compared to 37 °C (θ = 50° ± 1). Thus PNIPAAm film on the microgrooves exhibits responsive swelling with higher hydrophilicity at room temperature, which could be used to retrieve tissue constructs. The resulting tissue constructs were the same size as the grooves and could be used as modules in tissue fabrication. Given its ability to form and retrieve cell aggregates and its integration with standard microfabrication, PNIPAAm-coated PDMS templates may become useful for 3D cell culture applications in tissue engineering and drug discovery.