Successful reprogramming of epiblast stem cells by blocking nuclear localization of β-catenin.

Epiblast stem cells (EpiSCs) in mice and rats are primed pluripotent stem cells (PSCs). They barely contribute to chimeric embryos when injected into blastocysts. Reprogramming of EpiSCs to embryonic stem cell (ESC)-like cells (rESCs) may occur in response to LIF-STAT3 signaling; however, low reprogramming efficiency hampers potential use of rESCs in generating chimeras. Here, we describe dramatic improvement of conversion efficiency from primed to naive-like PSCs through upregulation of E-cadherin in the presence of the cytokine LIF. Analysis revealed that blocking nuclear localization of β-CATENIN with small-molecule inhibitors significantly enhances reprogramming efficiency of mouse EpiSCs. Although activation of Wnt/β-catenin signals has been thought desirable for maintenance of naive PSCs, this study provides the evidence that inhibition of nuclear translocation of β-CATENIN enhances conversion of mouse EpiSCs to naive-like PSCs (rESCs). This affords better understanding of gene regulatory circuits underlying pluripotency and reprogramming of PSCs

Alternatively activated macrophages promote pancreatic fibrosis in chronic pancreatitis.

Chronic pancreatitis (CP) is a progressive and irreversible inflammatory and fibrotic disease with no cure. Unlike acute pancreatitis (AP), we find that alternatively activated macrophages (AAMs) are dominant in mouse and human CP. AAMs are dependent on interleukin (IL)-4 and IL-13 signalling, and we show that mice lacking IL-4Rα, myeloid-specific IL-4Rα and IL-4/IL-13 were less susceptible to pancreatic fibrosis. Furthermore, we demonstrate that mouse and human pancreatic stellate cells (PSCs) are a source of IL-4/IL-13. Notably, we show that pharmacologic inhibition of IL-4/IL-13 in human ex vivo studies as well as in established mouse CP decreases pancreatic AAMs and fibrosis. We identify a critical role for macrophages in pancreatic fibrosis and in turn PSCs as important inducers of macrophage-alternative activation. Our study challenges and identifies pathways involved in crosstalk between macrophages and PSCs that can be targeted to reverse or halt pancreatic fibrosis progression

CXCL12/SDF-1 from perisynaptic Schwann cells promotes regeneration of injured motor axonterminals

The neuromuscular junction has retained through evolution the capacity to regenerate after damage, but little is known on the inter-cellular signals involved in its functional recovery from trauma, autoimmune attacks, or neurotoxins. We report here that CXCL12, also abbreviated as stromal-derived factor-1 (SDF-1), is produced specifically by perisynaptic Schwann cells following motor axon terminal degeneration induced by -latrotoxin. CXCL12 acts via binding to the neuronal CXCR4 receptor. A CXCL12-neutralizing antibody or a specific CXCR4 inhibitor strongly delays recovery from motor neuron degeneration invivo. Recombinant CXCL12 invivo accelerates neurotransmission rescue upon damage and very effectively stimulates the axon growth of spinal cord motor neurons invitro. These findings indicate that the CXCL12-CXCR4 axis plays an important role in the regeneration of the neuromuscular junction after motor axon injury. The present results have important implications in the effort to find therapeutics and protocols to improve recovery of function after different forms of motor axon terminal damage

ATRA mechanically reprograms pancreatic stellate cells to suppress matrix remodelling and inhibit cancer cell invasion

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a dismal survival rate. Persistent activation of pancreatic stellate cells (PSCs) can perturb the biomechanical homoeostasis of the tumour microenvironment to favour cancer cell invasion. Here we report that ATRA, an active metabolite of vitamin A, restores mechanical quiescence in PSCs via a mechanism involving a retinoic acid receptor beta (RAR-β)-dependent downregulation of actomyosin (MLC-2) contractility. We show that ATRA reduces the ability of PSCs to generate high traction forces and adapt to extracellular mechanical cues (mechanosensing), as well as suppresses force-mediated extracellular matrix remodelling to inhibit local cancer cell invasion in 3D organotypic models. Our findings implicate a RAR-β/MLC-2 pathway in peritumoural stromal remodelling and mechanosensory-driven activation of PSCs, and further suggest that mechanical reprogramming of PSCs with retinoic acid derivatives might be a viable alternative to stromal ablation strategies for the treatment of PDAC

The effects of age and ganglioside composition on the rate of motor nerve terminal regeneration following antibody-mediated injury in mice

Gangliosides are glycosphingolipids highly enriched in neural plasma membranes, where they mediate a diverse range of functions and can act as targets for auto-antibodies present in human immune-mediated neuropathy sera. The ensuing autoimmune injury results in axonal and motor nerve terminal (mNT) degeneration. Both aging and ganglioside-deficiency have been linked to impaired axonal regeneration. To assess the effects of age and ganglioside expression on mNT regeneration in an autoimmune injury paradigm, anti-ganglioside antibodies and complement were applied to young adult and aged mice wildtype (WT) mice, mice deficient in either b- and c-series (GD3sKO) or mice deficient in all complex gangliosides (GM2sKO). The extent of mNT injury and regeneration was assessed immediately or after 5 days, respectively. Depending on ganglioside expression and antibody-specificity, either a selective mNT injury or a combined injury of mNTs and neuromuscular glial cells was elicited. Immediately after induction of the injury, between 1.5% and 11.8% of neuromuscular junctions (NMJs) in the young adult groups exhibited healthy mNTs. Five days later, most NMJs, regardless of age and strain, had recovered their mNTs. No significant differences could be observed between young and aged WT and GM2sKO mice; aged GD3sKO showed a mildly impaired rate of mNT regeneration when compared with their younger counterparts. Comparable rates were observed between all strains in the young and the aged mice. In summary, the rate of mNT regeneration following anti-ganglioside antibody and complement-mediated injury does not differ majorly between young adult and aged mice irrespective of the expression of particular gangliosides

Loss of Pten causes tumor initiation following differentiation of murine pluripotent stem cells due to failed repression of Nanog.

Pluripotent stem cells (PSCs) hold significant promise in regenerative medicine due to their unlimited capacity for self-renewal and potential to differentiate into every cell type in the body. One major barrier to the use of PSCs is their potential risk for tumor initiation following differentiation and transplantation in vivo. In the current study we sought to evaluate the role of the tumor suppressor Pten in murine PSC neoplastic progression. Using eight functional assays that have previously been used to indicate PSC adaptation or transformation, Pten null embryonic stem cells (ESCs) failed to rate as significant in five of them. Instead, our data demonstrate that the loss of Pten causes the emergence of a small number of aggressive, teratoma-initiating embryonic carcinoma cells (ECCs) during differentiation in vitro, while the remaining 90-95% of differentiated cells are non-tumorigenic. Furthermore, our data show that the mechanism by which Pten null ECCs emerge in vitro and cause tumors in vivo is through increased survival and self-renewal, due to failed repression of the transcription factor Nanog

In Vivo Generation of Engraftable Murine Hematopoietic Stem Cells by Gfi1b, c-Fos, and Gata2 Overexpression within Teratoma.

Generation of hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) could potentially provide unlimited HSCs for clinical transplantation, a curative treatment for numerous blood diseases. However, to date, bona fide HSC generation has been largely unsuccessful in vitro. We have previously described proof of concept for in vivo HSC generation from PSCs via teratoma formation. However, our first-generation system was complex and the output low. Here, we further optimize this technology and demonstrate the following: (1) simplified HSC generation using transcription factor overexpression; (2) improved HSC output using c-Kit-deficient host mice, and (3) that teratomas can be transplanted and cryopreserved. We demonstrate that overexpression of Gfi1b, c-Fos, and Gata2, previously reported to transdifferentiate fibroblasts into hematopoietic progenitors in vitro, can induce long-term HSC formation in vivo. Our in vivo system provides a useful platform to investigate new strategies and re-evaluate existing strategies to generate HSCs and study HSC development