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.