Long-term repair of porcine articular cartilage using cryopreservable, clinically compatible human embryonic stem cell-derived chondrocytes.

Osteoarthritis (OA) impacts hundreds of millions of people worldwide, with those affected incurring significant physical and financial burdens. Injuries such as focal defects to the articular surface are a major contributing risk factor for the development of OA. Current cartilage repair strategies are moderately effective at reducing pain but often replace damaged tissue with biomechanically inferior fibrocartilage. Here we describe the development, transcriptomic ontogenetic characterization and quality assessment at the single cell level, as well as the scaled manufacturing of an allogeneic human pluripotent stem cell-derived articular chondrocyte formulation that exhibits long-term functional repair of porcine articular cartilage. These results define a new potential clinical paradigm for articular cartilage repair and mitigation of the associated risk of OA.

Mapping molecular landmarks of human skeletal ontogeny and pluripotent stem cell-derived articular chondrocytes.

Tissue-specific gene expression defines cellular identity and function, but knowledge of early human development is limited, hampering application of cell-based therapies. Here we profiled 5 distinct cell types at a single fetal stage, as well as chondrocytes at 4 stages in vivo and 2 stages during in vitro differentiation. Network analysis delineated five tissue-specific gene modules; these modules and chromatin state analysis defined broad similarities in gene expression during cartilage specification and maturation in vitro and in vivo, including early expression and progressive silencing of muscle- and bone-specific genes. Finally, ontogenetic analysis of freshly isolated and pluripotent stem cell-derived articular chondrocytes identified that integrin alpha 4 defines 2 subsets of functionally and molecularly distinct chondrocytes characterized by their gene expression, osteochondral potential in vitro and proliferative signature in vivo. These analyses provide new insight into human musculoskeletal development and provide an essential comparative resource for disease modeling and regenerative medicine.

Molecular characterization of physis tissue by RNA sequencing.

The physis is a well-established and anatomically distinct cartilaginous structure that is crucial for normal long-bone development and growth. Abnormalities in physis function are linked to growth plate disorders and other pediatric musculoskeletal diseases. Understanding the molecular pathways operative in the physis may permit development of regenerative therapies to complement surgically-based procedures that are the current standard of care for growth plate disorders. Here, we performed next generation RNA sequencing on mRNA isolated from human physis and other skeletal tissues (e.g., articular cartilage and bone; n = 7 for each tissue). We observed statistically significant enrichment of gene sets in the physis when compared to the other musculoskeletal tissues. Further analysis of these upregulated genes identified physis-specific networks of extracellular matrix proteins including collagens (COL2A1, COL6A1, COL9A1, COL14A1, COL16A1) and matrilins (MATN1, MATN2, MATN3), and signaling proteins in the WNT pathway (WNT10B, FZD1, FZD10, DKK2) or the FGF pathway (FGF10, FGFR4). Our results provide further insight into the gene expression networks that contribute to the physis' unique structural composition and regulatory signaling networks. Physis-specific expression profiles may guide ongoing initiatives in tissue engineering and cell-based therapies for treatment of growth plate disorders and growth modulation therapies. Furthermore, our findings provide new leads for therapeutic drug discovery that would permit future intervention through pharmacological rather than surgical strategies.

Drug-induced modulation of gp130 signalling prevents articular cartilage degeneration and promotes repair.

OBJECTIVE: Human adult articular cartilage (AC) has little capacity for repair, and joint surface injuries often result in osteoarthritis (OA), characterised by loss of matrix, hypertrophy and chondrocyte apoptosis. Inflammation mediated by interleukin (IL)-6 family cytokines has been identified as a critical driver of proarthritic changes in mouse and human joints, resulting in a feed-forward process driving expression of matrix degrading enzymes and IL-6 itself. Here we show that signalling through glycoprotein 130 (gp130), the common receptor for IL-6 family cytokines, can have both context-specific and cytokine-specific effects on articular chondrocytes and that a small molecule gp130 modulator can bias signalling towards anti-inflammatory and antidegenerative outputs. METHODS: High throughput screening of 170 000 compounds identified a small molecule gp130 modulator termed regulator of cartilage growth and differentiation (RCGD 423) that promotes atypical homodimeric signalling in the absence of cytokine ligands, driving transient increases in MYC and pSTAT3 while suppressing oncostatin M- and IL-6-mediated activation of ERK and NF-κB via direct competition for gp130 occupancy. RESULTS: This small molecule increased proliferation while reducing apoptosis and hypertrophic responses in adult chondrocytes in vitro. In a rat partial meniscectomy model, RCGD 423 greatly reduced chondrocyte hypertrophy, loss and degeneration while increasing chondrocyte proliferation beyond that observed in response to injury. Moreover, RCGD 423 improved cartilage healing in a rat full-thickness osteochondral defect model, increasing proliferation of mesenchymal cells in the defect and also inhibiting breakdown of cartilage matrix in de novo generated cartilage. CONCLUSION: These results identify a novel strategy for AC remediation via small molecule-mediated modulation of gp130 signalling.

The TEAD family transcription factor Scalloped regulates blood progenitor maintenance and proliferation in Drosophila through PDGF/VEGFR receptor (Pvr) signaling.

The Drosophila lymph gland is a well-characterized hematopoietic organ in which a population of multipotent stem-like progenitors is maintained by a combination of signals from different cellular populations within the organ. The lymph gland serves as an ideal model both for the interrogation of signaling mechanisms involved in progenitor maintenance as well as a tool for the identification of novel regulatory mechanisms in the highly conserved process of hematopoiesis. Here, we demonstrate a requirement for the TEAD transcription factor Scalloped in the maintenance and proliferation of hematopoietic progenitors. We have characterized a novel population of hemocytes in the early lymph gland identified by the expression of Hand, Scalloped, and the PVR ligand PVF2. In this unique population, we show that Scalloped maintains PVF2 expression, which is required for hemocyte proliferation and achievement of normal lymph gland size. We further demonstrate that STAT signaling marks actively proliferating hemocytes in the early lymph gland, and inhibition of this pathway causes decreased lymph gland growth similar to loss of Scalloped and PVF2, demonstrating a requirement for PVR/STAT signaling in the regulation of lymph gland size. Finally, we demonstrate that Scalloped regulates PVR expression and the maintenance of progenitors downstream of PVR/STAT/ADGF signaling. These findings further establish the role of the TEAD family transcription factors in the regulation of important signaling molecules, and expand our mechanistic insight into the balance between progenitor maintenance and proliferation required for the regulation of lymph gland homeostasis.

Yorkie and Scalloped signaling regulates Notch-dependent lineage specification during Drosophila hematopoiesis.

Cellular microenvironments established by the spatial and temporal expression of specific signaling molecules are critical for both the maintenance and lineage-specific differentiation of progenitor cells. In Drosophila, a population of hematopoietic progenitors, or prohemocytes, within the larval lymph gland gives rise to three mature cell types: plasmatocytes, lamellocytes, and crystal cells. Removal of the secreted signaling molecules Hedgehog and PVF1 from the posterior signaling center (PSC), which acts as a niche, leads to a loss of progenitors and complete differentiation of the lymph gland. Here, we characterize a novel population of signaling cells within the lymph gland, distinct from the PSC, that are required for lineage-specific differentiation of crystal cells. We provide evidence that Yorkie and Scalloped, the Drosophila homologs of YAP and TEAD, are required in lineage-specifying cells to regulate expression of Serrate, the Notch ligand responsible for the initiation of the crystal cell differentiation program. Genetic manipulation of yorkie and scalloped in the lymph gland specifically alters Serrate expression and crystal cell differentiation. Furthermore, Serrate expression in lineage-specifying cells is eliminated in the lymph gland upon the immune response induced by wasp parasitization to ensure the proper differentiation of lamellocytes at the expense of crystal cells. These findings expand the roles for Yorkie/Scalloped beyond growth to encompass specific cell-fate determination in the context of blood development. Similar regulatory functions may extend to their homologs in vertebrate progenitor cell niches that are required for specifying cell fate.

Kicking it up a Notch for the best in show: Scalloped leads Yorkie into the haematopoietic arena.

Maintenance and differentiation of progenitor cells is essential for proper organ development and adaptation to environmental stress and injury. In Drosophila melanogaster, the haematopietic system serves as an ideal model for interrogating the function of signaling pathways required for progenitor maintenance and cell fate determination. Here we focus on the role of the Hippo pathway effectors Yorkie and Scalloped in mediating and facilitating Notch signaling-mediated lineage specification in the lymph gland, the primary center for haematopoiesis within the developing larva. We discuss the regulatory mechanisms which promote Notch activity during normal haematopoiesis and its modulation during immune challenge conditions. We provide additional evidence establishing the hierarchy of signaling events during crystal cell formation, highlighting the relationship between Yorkie, Scalloped and Lozenge, while expanding on the role of Yorkie in promoting hemocyte survival and the developmental regulation of Notch and its ligand, Serrate, within the lymph gland. Finally, we propose additional areas of exploration that may provide mechanistic insight into the environmental and non-cell autonomous regulation of cell fate in the blood system.