Induced pluripotent stem cells (iPSC) hold tremendous potential for personalized cell-based repair strategies to treat musculoskeletal disorders. To establish human iPSCs as a potential source of viable chondroprogenitors for articular cartilage repair, we assessed the in vitro chondrogenic potential of the pluripotent population versus an iPSC-derived mesenchymal-like progenitor population. We found the direct plating of undifferentiated iPSCs into high-density micromass cultures in the presence of BMP-2 promoted chondrogenic differentiation, however these conditions resulted in a mixed population of cells resembling the phenotype of articular cartilage, transient cartilage, and fibrocartilage. The progenitor cells derived from human iPSCs exhibited immunophenotypic features of mesenchymal stem cells (MSCs) and developed along multiple mesenchymal lineages, including osteoblasts, adipocytes, and chondrocytes in vitro. The data indicate the derivation of a mesenchymal stem cell population from human iPSCs is necessary to limit culture heterogeneity as well as chondrocyte maturation in the differentiated progeny. Moreover, as compared to pellet culture differentiation, BMP-2 treatment of iPSC-derived MSC-like (iPSC-MSC) micromass cultures resulted in a phenotype more typical of articular chondrocytes, characterized by the enrichment of cartilage-specific type II collagen (Col2a1), decreased expression of type I collagen (Col1a1) as well as lack of chondrocyte hypertrophy. These studies represent a first step toward identifying the most suitable iPSC progeny for developing cell-based approaches to repair joint cartilage damage.

We characterized the interaction of amylin with heparin fragments of defined length, which model the glycosaminoglycan chains associated with amyloid deposits found in type 2 diabetes. Binding of heparin fragments to the positively charged N-terminal half of monomeric amylin depends on the concentration of negatively charged saccharides but is independent of oligosaccharide length. By contrast, amylin fibrillogenesis has a sigmoidal dependence on heparin fragment length, with an enhancement observed for oligosaccharides longer than four monomers and a leveling off of effects beyond 12 monomers. The length dependence suggests that the negatively charged helical structure of heparin electrostatically complements the positively charged surface of the fibrillar amylin cross-␤ structure. Fluorescence resonance energy transfer and total internal reflection fluorescence microscopy experiments indicate that heparin associates with amylin fibrils, rather than enhancing fibrillogenesis catalytically. Short heparin fragments containing two-or eight-saccharide monomers protect against amylin cytotoxicity toward a MIN6 mouse cell model of pancreatic ␤-cells.Type 2 diabetes accounts for ϳ90% of adult diabetes. The disease currently affects 200 million people worldwide, and its incidence is projected to rise to 300 million by 2025 (1). Like many complex diseases, type 2 diabetes has multifactorial origins (1-4). The disease is characterized by insulin resistance, which causes the pancreas to synthesize more of the hormones insulin and amylin (5). The late stages of the disease are associated with ␤-cell dysfunction and a loss of ␤-cell mass (3, 5).Amylin (also known as islet amyloid polypeptide) is a 37-residue (4-KDa) endocrine hormone that is synthesized by pancreatic ␤-cells and co-secreted with insulin. In its normal state, amylin works together with insulin to control blood sugar (5). Additional amylin functions include suppressing appetite, slowing down the emptying of the stomach, and inhibiting glucagon secretion (5, 6). Like some 30 other polypeptides associated with human amyloid pathologies (6), amylin can undergo aggregative misfolding into fibrils with a cross-␤ structure (5,7,8). Although it is still uncertain whether fibrils or soluble oligomeric precursors are responsible for adverse affects (8 -12), extracellular amylin aggregates have been implicated in the destruction of pancreatic ␤-cells during the progression of type 2 diabetes (5, 9, 13). Amylin is the main component of fibrillar amyloid deposits found in the interstitial fluid between pancreatic islet cells of type 2 diabetes patients tested post-mortem (3,5,14), and extracellular aggregates of amylin are toxic when added to cultures of ␤-cells (9, 13). Overexpression of human amylin has been found to correlate with ␤-cell apoptosis and diabetes-like symptoms in several transgenic mouse and rat models whose endogenous amylin does not fibrillize (15). Genetic evidence that amylin is involved in pathology comes from the familial S20G mutation, which leads...

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The success of cell‐based therapies to restore joint cartilage requires an optimal source of reparative progenitor cells and tight control of their differentiation into a permanent cartilage phenotype. Bone morphogenetic protein 2 (BMP‐2) has been extensively shown to promote mesenchymal cell differentiation into chondrocytes in vitro and in vivo. Conversely, developmental studies have demonstrated decreased chondrocyte maturation by Wingless‐Type MMTV Integration Site Family, Member 5A (Wnt5a). Thus, we hypothesized that treatment of human embryonic stem cell (hESC)‐derived chondroprogenitors with BMP‐2 followed by Wnt5a may control the maturational progression of these cells into a hyaline‐like chondrocyte phenotype. We examined the effects of sustained exposure of hESC‐derived mesenchymal‐like progenitors to recombinant Wnt5a or BMP‐2 in vitro. Our data indicate that BMP‐2 promoted a strong chondrogenic response leading to terminal maturation, whereas recombinant Wnt5a induced a mild chondrogenic response without promoting hypertrophy. Moreover, Wnt5a suppressed BMP‐2‐mediated chondrocyte maturation, preventing the formation of fibrocartilaginous tissue in high‐density cultures treated sequentially with BMP‐2 and Wnt5a. Implantation of scaffoldless pellets of hESC‐derived chondroprogenitors pretreated with BMP‐2 followed by Wnt5a into rat chondral defects induced an articular‐like phenotype in vivo. Together, the data establish a novel role for Wnt5a in controlling the progression from multipotency into an articular‐like cartilage phenotype in vitro and in vivo. Stem Cells Translational Medicine 2017;6:40–50

Intestinal organoids are multicellular crypt-like structures that can be derived from adult intestinal stem cells (ISCs), embryonic stem cells (ESCs) or induced pluripotent stem cells (IPSCs). Here we show that intestinal organoids generated from mouse ESCs were enriched in ISCs and early progenitors. Treatment of these organoids with a γ-secretase inhibitor increased Math1 and decreased Hes1 expression, indicating Notch signaling regulates ISC differentiation in these organoids. Lgr5 and Tert positive ISCs constituted approximately 10% and 20% of the organoids. As found in native tissue, Lgr5 and Tert expressing cells resolved into two discreet populations, which were stable over time. Intestinal organoids derived from cancer-prone Apc(Min/+) mice showed similar numbers of ISCs, but had reduced Math1 expression, indicating a suppressed secretory cell differentiation potential (as found in intestinal tissue). Apc(Min/+) organoids were used to screen epigenetically active compounds for those that increased Math1 expression and organoid differentiation (including HDAC inhibitors, Sirtuin (SIRT) modulators and methyltransferase inhibitors). Broad-spectrum HDAC inhibitors increased both Math1 and Muc2 expression, indicating an ability to promote the suppressed secretory cell differentiation pathway. Other epigenetic compounds had a diverse impact on cell differentiation, with a strong negative correlation between those that activated the secretory marker Muc2 and those that activated the absorptive cell marker Fabp2. These data show that ESC-derived intestinal organoids can be derived in large numbers, contain distinct ISC types and can be used to screen for agents that promote cell differentiation through different lineage pathways.