Jason D. Gibson

Efficient differentiation of human iPSC‐derived mesenchymal stem cells to chondroprogenitor cells

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. J. Cell. Biochem. 114: 480–490, 2013.

Mechanism of Amylin Fibrillization Enhancement by Heparin

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-β 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 β-cells.

Intestinal lineage commitment of embryonic stem cells.

Generating lineage-committed intestinal stem cells from embryonic stem cells (ESCs) could provide a tractable experimental system for understanding intestinal differentiation pathways and may ultimately provide cells for regenerating damaged intestinal tissue. We tested a two-step differentiation procedure in which ESCs were first cultured with activin A to favor formation of definitive endoderm, and then treated with fibroblast-conditioned medium with or without Wnt3A. The definitive endoderm expressed a number of genes associated with gut-tube development through mouse embryonic day 8.5 (Sox17, Foxa2, and Gata4 expressed and Id2 silent). The intestinal stem cell marker Lgr5 gene was also activated in the endodermal cells, whereas the Msi1, Ephb2, and Dcamkl1 intestinal stem cell markers were not. Exposure of the endoderm to fibroblast-conditioned medium with Wnt3A resulted in the activation of Id2, the remaining intestinal stem cell markers and the later gut markers Cdx2, Fabp2, and Muc2. Interestingly, genes associated with distal gut-associated mesoderm (Foxf2, Hlx, and Hoxd8) were also simulated by Wnt3A. The two-step differentiation protocol generated gut bodies with crypt-like structures that included regions of Lgr5-expressing proliferating cells and regions of cell differentiation. These gut bodies also had a smooth muscle component and some underwent peristaltic movement. The ability of the definitive endoderm to differentiate into intestinal epithelium was supported by the vivo engraftment of these cells into mouse colonic mucosa. These findings demonstrate that definitive endoderm derived from ESCs can carry out intestinal cell differentiation pathways and may provide cells to restore damaged intestinal tissue.

Development of intestinal organoids as tissue surrogates: Cell composition and the Epigenetic control of differentiation

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 ApcMin/+ mice showed similar numbers of ISCs, but had reduced Math1 expression, indicating a suppressed secretory cell differentiation potential (as found in intestinal tissue). ApcMin/+ 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.

Regeneration of Articular Cartilage by Human ESC-Derived Mesenchymal Progenitors Treated Sequentially With BMP-2 and Wnt5a

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

SCLD: a stem cell lineage database for the annotation of cell types and developmental lineages

Stem cell biology has experienced explosive growth over the past decade as researchers attempt to generate therapeutically relevant cell types in the laboratory. Recapitulation of endogenous developmental trajectories is a dominant paradigm in the design of directed differentiation protocols, and attempts to guide stem cell differentiation are often based explicitly on knowledge of in vivo development. Therefore, when designing protocols, stem cell biologists rely heavily upon information including (i) cell type-specific gene expression profiles, (ii) anatomical and developmental relationships between cells and tissues and (iii) signals important for progression from progenitors to target cell types. Here, we present the Stem Cell Lineage Database (SCLD) (http://scld.mcb.uconn.edu) that aims to unify this information into a single resource where users can easily store and access information about cell type gene expression, cell lineage maps and stem cell differentiation protocols for both human and mouse stem cells and endogenous developmental lineages. By establishing the SCLD, we provide scientists with a centralized location to organize access and share data, dispute and resolve contentious relationships between cell types and within lineages, uncover discriminating cell type marker panels and design directed differentiation protocols.

Derivation and Chondrogenic Commitment of Human Embryonic Stem Cell-Derived Mesenchymal Progenitors

The induction of human embryonic stem cells to a mesenchymal-like progenitor population constitutes a developmentally relevant approach for efficient directed differentiation of human embryonic stem (hES) cells to the chondrogenic lineage. The initial enrichment of a hemangioblast intermediate has been shown to yield a replenishable population of highly purified progenitor cells that exhibit the typical mesenchymal stem cell (MSC) surface markers as well as the capacity for multilineage differentiation to bone, fat, and cartilage. Herein, we provide detailed methodologies for the derivation and characterization of potent mesenchymal-like progenitors from hES cells and describe in vitro assays for bone morphogenetic protein (BMP)-2-mediated differentiation to the chondrogenic lineage.

Aberrant expression of Twist1 in diseased articular cartilage and a potential role in the modulation of osteoarthritis severity

The bHLH transcription factor Twist1 has emerged as a negative regulator of chondrogenesis in skeletal progenitor cells and as an inhibitor of maturation in growth plate chondrocytes. However, its role in articular cartilage remains obscure. Here we examine Twist1 expression during re-differentiation of expanded human articular chondrocytes, the distribution of Twist1 proteins in normal versus OA human articular cartilage, and its role in modulating OA development in mice. High levels of Twist1 transcripts were detected by qPCR analyses of expanded de-differentiated human articular chondrocytes that had acquired mesenchymal-like features. The induction of hallmark cartilage genes by Bmp-2 mediated chondrogenic differentiation was paralleled by the dramatic suppression of Twist1 in vitro. In normal human articular cartilage, Twist1-expressing chondrocytes were most abundant in the superficial zone with little to no expression in the middle and deep zones. However, our analyses revealed a higher proportion of deep zone articular chondrocytes expressing Twist1 in human OA cartilage as compared to normal articular cartilage. Moreover, Twist1 expression was prominent within proliferative cell clusters near fissure sites in more severely affected OA samples. To assess the role of Twist1 in OA pathophysiology, we subjected wild type mice and transgenic mice with gain of Twist1 function in cartilage to surgical destabilization of the medial meniscus. At 12 weeks post-surgery, micro-CT and histological analyses revealed attenuation of the OA phenotype in Twist1 transgenic mice compared to wild type mice. Collectively, the data reveal a role for Twist in articular cartilage maintenance and the attenuation of cartilage degeneration.