Generalized arterial calcification of infancy (GACI) is an autosomal recessive disease, characterized by widespread calcification of the cardiovascular system, and often results in death before the first year of life. The ENPP1 gene is mutated in most GACI cases, and currently there is no effective treatment. The asj-2J mutant mouse, carrying a large deletion/insertion mutation in the ENPP1 gene, has been characterized as a model for GACI, with previous studies focusing on mineralization of skin and vascular tissues. In this study, we examined the ectopic mineralization of cartilage, collagen-rich tendons and ligaments as well as skeletal mineralization in this mouse model. Standard histology was performed on soft connective tissues, such as ear (elastic cartilage) and trachea (hyaline cartilage). A novel cryo-histological method was used to assess ectopic mineralization of articular cartilage and fibrocartilage as well as tendon and ligaments at bone insertion sites in non-decalcified sections. Ectopic mineralization was seen in the ear, trachea, cartilage, tendons and ligaments in homozygous asj-2J mice at 12 weeks of age. Bone formation and resorption were analyzed via alkaline phosphate (AP) and tartrate-resistant acid phosphatase (TRAP) enzyme activity, respectively. Immature osteophytes with AP and TRAP activities were evident as early as 6 weeks of age. Increased bone formation rate in these mice was also indicated by the significant increase in AP activity in asj-2J mouse serum compared to wild-type. These data highlight the important role of ENPP1 in regulating calcification of soft and skeletal tissues.

This manuscript reports the characterization of molecularly self-assembled collagen nanofibers on a natural polymeric microporous structure and their ability to support stem cell differentiationin vitroand host tissue responsein vivo.

Purpose: There has been great interest in deciphering how articular cartilage acquires its multiple, distinct zones, including the superficial zone that produces lubricants and may contain stem/progenitor cells. We previously demonstrated that a population Gdf-5 expressing mesenchymal cells at presumptive limb joint sites gives rise to multiple joint tissues over time, including the articular cartilage, synovial lining, ligaments, and tendon entheses. However, remaining questions include whether unique subpopulations of cells exist within this population of joint progenitor cells, if these cells selectively participate in development of specific joint tissues, and what roles they may have in postnatal joint maturation and repair. We have utilized novel lineage tracing and imaging techniques to address these and other related questions. Methods: We evaluated progenies of Gdf-5 expressing lineage cells by crossing female Gdf-5Cre mice with male ROSA-zsGreen (single color) or ROSA-Confetti (multi-color) reporter mice. After Cre-mediated recombination in ROSA-Confetti mice, each cell and its progeny are distinctively labeled with one of four unique colors (GFP, YFP, RFP and CFP), permitting discrimination of unique clones within the same tissue. During embryonic development, Dkk-3 expression patterns broadly overlap those of Gdf-5. We created a novel inducible BAC transgenic Dkk3CreER-T2 mouse line to permit selective labeling of joint progenitor cells. Tamoxifen was administered intraperitoneally to timed-pregnant female Dkk3CreER-T2 mice mated to male ROSA-zsGreen mice. To evaluate the acute response to cartilage injury, focal chondral defects (FCDs) were created in the femur of adult mice, and limbs were collected 1 week after surgery. Imaging of limb sections and whole mount samples was conducted by confocal and multiphoton microscopy. Results: At birth, cells and progenies with a Gdf-5 expressing lineage were visible throughout all joint tissues in Gdf5Cre;ROSAzsGreen knees (Fig. 1A). Fluorescently labeled cells remained throughout all layers of the articular cartilage at 2 months of age (Fig. 1B). Interestingly, the tibial articular cartilage of Gdf-5Cre;ROSA-Confetti displayed a mosaic distribution of uniquely colored clones at 2 months of age (Fig.1 C-D). Three-dimensional analysis of the intact tibial plateau revealed that expansion of clones occurred within limited, defined domains (Fig. 1D). These clones were also limited in size - suggesting limited cell proliferation over time. Although dynamic with development, stage-specific expression of Dkk-3 at E17.5 (Fig. 2A) was accurately captured by tamoxifen administration to Dkk3CreER-T2 mice at E17.5 (Fig. 2B). Dkk-3 lineage cells/progeny remained in joint tissues postnatally (Fig. 2C-F), and, notably maintained expression of MSC markers including CD-44 in the synovium (Fig. 2D). When FCDs were created in these mice at 2M of age, ROSAzsGreen labeled cells were found within defect sites (Fig. 2F, arrowheads) during acute response to injury. Conclusions: Our cell lineage tracing data reveal that progenies of embryonic joint site-associated cell populations are long-lived and persist in postnatal joints, highlighting their role in morphogenesis, maintenance and repair of joint tissues. While previous research suggested that articular cartilage growth occurs mainly through appositional expansion from a superficial progenitor cell population, our data suggest that postnatal growth may also occur through a process of local cell intercalation. These data also demonstrate the exciting potential of these novel cell tracing and imaging techniques for studying spatiotemporal gene activation and function during postnatal joint growth and morphogenesis.

ObjectiveA major challenge to understanding osteoarthritis (OA) pathology is identifying the cellular events that precede the onset of cartilage damage. The objective of this study is to determine the effect of joint destabilization on early changes to fibrocartilage in the joint.Design/MethodsThe anterior cruciate ligament was transected in collagen reporter mice (Col1CFP and ColXRFP). Mineralization labels were given every 2 weeks to measure new mineralized cartilage apposition. Novel fluorescent histology of mineralized tissue was used to characterize the changes in fibrocartilage at 2 and 4 weeks post-injury.ResultsChanges in fibrocartilaginous structures of the joint occur as early as 2 weeks after injury and are well developed by 4 weeks. The alterations are seen in multiple entheses and in the medial surface of the femoral and tibial condyles. In the responding entheses, mineral apposition towards the ligament midsubstance results in thickening of the mineralize fibrocartilage. These changes are associated with increases in ColX-RFP, Col1-CFP reporter activity and alkaline phosphatase enzyme activity. Mineral apposition also occurs in the fibrocartilage of the non-articular regions of the medial condyles by 2 weeks and develops into osteophytes by 4 weeks post-injury. An unexpected observation is punctate expression of tartrate resistant acid phosphatase activity in unmineralized fibrochondrocytes adjacent to active appositional mineralization.DiscussionThese observations suggest that fibrocartilage activates prior to degradation of the articular cartilage. Thus clinical and histological imaging of fibrocartilage may be an earlier indicator of disease initiation and may indicate a more appropriate time to start preventative treatment.

Summary Objectives The generation of transgenic mice expressing green fluorescent proteins (GFPs) has greatly aided our understanding of the development of connective tissues such as bone and cartilage. Perturbation of a biological system such as the temporomandibular joint (TMJ) within its adaptive remodeling capacity is particularly useful in analyzing cellular lineage progression. The objectives of this study were to determine: (i) if GFP reporters expressed in the TMJ indicate the different stages of cell maturation in fibrocartilage and (ii) how mechanical loading affects cellular response in different regions of the cartilage. Design/methods Four-week-old transgenic mice harboring combinations of fluorescent reporters (Dkk3-eGFP, Col1a1(3.6 kb)-GFPcyan, Col1a1(3.6 kb)-GFPtpz, Col2a1-GFPcyan, and Col10a1-RFPcherry) were used to analyze the expression pattern of transgenes in the mandibular condylar cartilage (MCC). To study the effect of TMJ loading, animals were subjected to forced mouth opening with custom springs exerting 50 g force for 1 h/day for 5 days. Dynamic mineralization and cellular proliferation (EdU-labeling) were assessed in loaded vs control mice. Results Dkk3 expression was seen in the superficial zone of the MCC, followed by Col1 in the cartilage zone, Col2 in the prehypertrophic zone, and Col10 in the hypertrophic zone at and below the tidemark. TMJ loading increased expression of the GFP reporters and EdU-labeling of cells in the cartilage, resulting in a thickness increase of all layers of the cartilage. In addition, mineral apposition increased resulting in Col10 expression by unmineralized cells above the tidemark. Conclusion The TMJ responded to static loading by forming thicker cartilage through adaptive remodeling.

AIM:Mustn1 has been implicated in myofusion as well as skeletal muscle growth and repair; however, the exact role and spatio-temporal expression of Mustn1 have yet to be fully defined.METHODS:Transgenic mice were generated with a 1512-bp sequence of the Mustn1 promoter directing the expression of GFP (Mustn1(PRO) -GFP). These mice were used to investigate the spatio-temporal expression of Mustn1(PRO) -GFP during skeletal muscle development and adult skeletal muscle repair, as well as various phases of the satellite cell lifespan (i.e. quiescence, activation, proliferation, differentiation).RESULTS:Mustn1(PRO) -GFP expression was observed within somites at embryonic day 12 and developing skeletal muscles at embryonic day 15 and 18. While uninjured adult tibialis anterior muscle displayed no detectable Mustn1(PRO) -GFP expression, cardiotoxin injury robustly elevated Mustn1(PRO) -GFP expression at 3 days post-injury with decreasing levels observed at 5 days and minimal, focal expression seen at 10 days. The expression of Mustn1(PRO) -GFP at 3 days post-injury consistently overlaid with MyoD although the strongest expression of Mustn1(PRO) -GFP was noted in newly formed myotubes that were expressing minimal levels of MyoD. By 5 days post-injury, Mustn1(PRO) -GFP overlaid in all myotubes expressing myogenin although cells were present expressing Mustn1(PRO) -GFP alone. The expression patterns of Mustn1(PRO) -GFP in regenerating muscle preceded the expression of desmin throughout the regenerative time course consistent with Mustn1 being upstream of this myogenic protein. Further, quiescent satellite cells located on freshly isolated, single myofibers rarely expressed Mustn1(PRO) -GFP, but within 24 h of isolation, all activated satellite cells expressed Mustn1(PRO) -GFP. Expression of Mustn1(PRO) -GFP in primary myoblasts diminished with prolonged time in proliferation media. However, in response to serum withdrawal, the expression of Mustn1(PRO) -GFP increased during myofusion (day 2) followed by declining expression thereafter.CONCLUSION:Mustn1(PRO) -GFP is expressed in activated satellite cells and myoblasts but continued time in proliferation media diminished Mustn1(PRO) -GFP expression. However, myoblasts exposed to serum withdrawal increased Mustn1(PRO) -GFP expression consistent with its demonstrated role in myofusion. The in vivo expression pattern of Mustn1 observed in regenerating and developing skeletal muscle is consistent with its presence in satellite cells and its critical role in myofusion.

Learning Outcome 1: The origins and characteristics of ESCs and iPSCs. Learning Outcome 2: How to control pluripotent stem cell differentiation to direct their development along specific lineages. Learning Outcome 3: The power of pluripotent stem cell technology to study development, disease mechanisms and therapy. Abstract: Recent breakthroughs in the stem cell biology field have demonstrated the potential of pluripotent Embryonic Stem Cells (ESCs) to generate any cell type in the body. This technology has been underpinned by advances in elucidating the regulatory networks involved in stemcell self-renewal andpluripotency, but also by insights provided by developmental biology regarding the transcriptional regulators and signaling pathways that set up the body plan and control organogenesis in the embryo. Efficient ESC differentiation protocols have been established, and continue to be modified and improved, that model mammalian development by recapitulating gastrulation in vitro through the induction of ectoderm, mesoderm and endoderm germ layers, and subsequently directing their further differentiation along their respective downstream lineages. The field of pluripotent stem cell biology has also been pushed to the forefront of biomedical researchby the important discovery of reprogramming somatic cells to anESClike state, generating induced Pluripotent Stem cells (iPSCs). The ability to reprogram cells has offered the unique opportunity to generate patient-specific iPSCs, and combine this with lineage-specific differentiation protocols to further model human disease in a dish and understand disease mechanisms. Taken together, pluripotent stem cell technology has provided the unique tools to not only model mammalian development, but also to generate functional cell types and tissues in vitro that can be used for drug discovery, for gaining insights into disease, as well as genetic rescue and potential autologous cell-based regenerative and replacement therapies. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.