Murine iPSC-Loaded Scaffold Grafts Improve Bone Regeneration in Critical-Size Bone Defects.

In certain situations, bones do not heal completely after fracturing. One of these situations is a critical-size bone defect where the bone cannot heal spontaneously. In such a case, complex fracture treatment over a long period of time is required, which carries a relevant risk of complications. The common methods used, such as autologous and allogeneic grafts, do not always lead to successful treatment results. Current approaches to increasing bone formation to bridge the gap include the application of stem cells on the fracture side. While most studies investigated the use of mesenchymal stromal cells, less evidence exists about induced pluripotent stem cells (iPSC). In this study, we investigated the potential of mouse iPSC-loaded scaffolds and decellularized scaffolds containing extracellular matrix from iPSCs for treating critical-size bone defects in a mouse model. In vitro differentiation followed by Alizarin Red staining and quantitative reverse transcription polymerase chain reaction confirmed the osteogenic differentiation potential of the iPSCs lines. Subsequently, an in vivo trial using a mouse model (n = 12) for critical-size bone defect was conducted, in which a PLGA/aCaP osteoconductive scaffold was transplanted into the bone defect for 9 weeks. Three groups (each n = 4) were defined as (1) osteoconductive scaffold only (control), (2) iPSC-derived extracellular matrix seeded on a scaffold and (3) iPSC seeded on a scaffold. Micro-CT and histological analysis show that iPSCs grafted onto an osteoconductive scaffold followed by induction of osteogenic differentiation resulted in significantly higher bone volume 9 weeks after implantation than an osteoconductive scaffold alone. Transplantation of iPSC-seeded PLGA/aCaP scaffolds may improve bone regeneration in critical-size bone defects in mice.

Artd1/Parp1 regulates reprogramming by transcriptional regulation of Fgf4 via Sox2 ADP-ribosylation.

The recently established reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by Takahashi and Yamanaka represents a valuable tool for future therapeutic applications. To date, the mechanisms underlying this process are still largely unknown. In particular, the mechanisms how the Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc) directly drive reprogramming and which additional components are involved are still not yet understood. In this study, we aimed at analyzing the role of ADP-ribosyltransferase diphtheria toxin-like one (Artd1; formerly called poly(ADP-ribose) polymerase 1 [Parp1]) during reprogramming. We found that poly(ADP-ribosylation) (PARylation) of the reprogramming factor Sox2 by Artd1 plays an important role during the first days upon transduction with the reprogramming factors. A process that happens before Artd1 in conjunction with 10-11 translocation-2 (Tet2) mediates the histone modifications necessary for the establishment of an activated chromatin state at pluripotency loci (e.g., Nanog and Essrb) [Nature 2012;488:652-655]. Wild-type (WT) fibroblasts treated with an Artd1 inhibitor as well as fibroblasts deficient for Artd1 (Artd1-/-) show strongly decreased reprogramming capacity. Our data indicate that Artd1-mediated PARylation of Sox2 favors its binding to the fibroblast growth factor 4 (Fgf4) enhancer, thereby activating Fgf4 expression. The importance of Fgf4 during the first 4 days upon initiation of reprogramming was also highlighted by the observation that exogenous addition of Fgf4 was sufficient to restore the reprogramming capacity of Artd1-/- fibroblast to WT levels. In conclusion, our data clearly show that the interaction between Artd1 and Sox2 is crucial for the first steps of the reprogramming process and that early expression of Fgf4 (day 2 to day 4) is an essential component for the successful generation of iPSCs.

Human serine protease HTRA1 positively regulates osteogenesis of human bone marrow-derived mesenchymal stem cells and mineralization of differentiating bone-forming cells through the modulation of extracellular matrix protein.

Mammalian high-temperature requirement serine protease A1 (HTRA1) is a secreted member of the trypsin family of serine proteases which can degrade a variety of bone matrix proteins and as such has been implicated in musculoskeletal development. In this study, we have investigated the role of HTRA1 in mesenchymal stem cell (MSC) osteogenesis and suggest a potential mechanism through which it controls matrix mineralization by differentiating bone-forming cells. Osteogenic induction resulted in a significant elevation in the expression and secretion of HTRA1 in MSCs isolated from human bone marrow-derived MSCs (hBMSCs), mouse adipose-derived stromal cells (mASCs), and mouse embryonic stem cells. Recombinant HTRA1 enhanced the osteogenesis of hBMSCs as evidenced by significant changes in several osteogenic markers including integrin-binding sialoprotein (IBSP), bone morphogenetic protein 5 (BMP5), and sclerostin, and promoted matrix mineralization in differentiating bone-forming osteoblasts. These stimulatory effects were not observed with proteolytically inactive HTRA1 and were abolished by small interfering RNA against HTRA1. Moreover, loss of HTRA1 function resulted in enhanced adipogenesis of hBMSCs. HTRA1 Immunofluorescence studies showed colocalization of HTRA1 with IBSP protein in osteogenic mASC spheroid cultures and was confirmed as being a newly identified HTRA1 substrate in cell cultures and in proteolytic enzyme assays. A role for HTRA1 in bone regeneration in vivo was also alluded to in bone fracture repair studies where HTRA1 was found localized predominantly to areas of new bone formation in association with IBSP. These data therefore implicate HTRA1 as having a central role in osteogenesis through modification of proteins within the extracellular matrix.

Pramel7 mediates LIF/STAT3-dependent self-renewal in embryonic stem cells.

A unique and complex signaling network allows ESCs to undergo extended proliferation in vitro, while maintaining their capacity for multilineage differentiation. Genuine ESC identity can only be maintained when both self-renewal and suppression of differentiation are active and balanced. Here, we identify Pramel7 (preferentially expressed antigen in melanoma-like 7) as a novel factor crucial for maintenance of pluripotency and leukemia inhibitory factor (LIF)-mediated self-renewal in ESCs. In vivo, Pramel7 expression was exclusively found in the pluripotent pools of cells, namely, the central part of the morula and the inner cell mass of the blastocyst. Ablation of Pramel7 induced ESC differentiation, whereas its overexpression was sufficient to support long-term self-renewal in the absence of exogenous LIF. Furthermore, Pramel7 overexpression suppressed differentiation in ESCs in vitro and in vivo. This process was reversible, as on transgene excision cells reverted to a LIF-dependent state and regained their capacity to participate in the formation of chimeric mice. Molecularly, LIF directly controls Pramel7 expression, involving both STAT3-dependent transcriptional regulation and PI3K-dependent phosphorylation of glycogen synthase kinase 3ß. Pramel7 expression in turn confers constitutive self-renewal and prevents differentiation through inactivation of extracellular signal-regulated kinase phosphorylation. Accordingly, knockdown of Pramel7 promotes ESC differentiation in presence of LIF and even on forced STAT3-activation. Thus, Pramel7 represents a central and essential factor in the signaling network regulating pluripotency and self-renewal in ESCs.

Does toe clipping for genotyping interfere with later-in-life nociception in mice?

INTRODUCTION: Genetically modified mice are widely used in studies on human and animal physiology and pharmacology, including pain research. The experimental design usually includes comparisons of genetically modified mice with wild-type littermates, requiring biopsy material for genotyping and methods for unequivocal identification of individual mice. Ethical standards and, in some countries, legislation require that both needs are reached with a single procedure. Clipping of the most distal phalanx of up to two toes per paw (toe clipping) is the favored procedure in most research fields, but it may be problematic in sensory physiology and pain research. OBJECTIVES: To systematically investigate whether toe-clipping influences later-in-life nociceptive sensitivity or the susceptibility to neuropathic or inflammatory hyperalgesia. METHODS: We tested in male mice whether the clipping of 2 toes of a hind paw influences nociceptive sensitivities to noxious heat or cold, or to mechanical stimulation under baseline conditions, after peripheral nerve injury (chronic constriction of the sciatic nerve) or during peripheral inflammation induced by subcutaneous zymosan A injection. We tested not only for the presence of significant differences but also specifically addressed bioequivalence using the 2 one-sided t test procedure. We chose a threshold of 25% variation of the control value for nonequivalence, which is usually taken as a threshold for biological relevance in pain tests. RESULTS: Using this value, we found that for all conditions (non-neuropathic and non-inflamed, neuropathic and inflamed), nociceptive sensitivities significantly fell within the equivalence bounds of the non-toe-clipped control mice. CONCLUSIONS: These results suggest that toe clipping does not have long-term effects on nociceptive sensitivities and does not alter the susceptibility of male mice to neuropathic or inflammatory hyperalgesia.

Corrigendum: Pramel7 mediates ground-state pluripotency through proteasomal-epigenetic combined pathways.

This corrects the article DOI: 10.1038/ncb3554.

Pramel7 mediates ground-state pluripotency through proteasomal-epigenetic combined pathways.

Naive pluripotency is established in preimplantation epiblast. Embryonic stem cells (ESCs) represent the immortalization of naive pluripotency. 2i culture has optimized this state, leading to a gene signature and DNA hypomethylation closely comparable to preimplantation epiblast, the developmental ground state. Here we show that Pramel7 (PRAME-like 7), a protein highly expressed in the inner cell mass (ICM) but expressed at low levels in ESCs, targets for proteasomal degradation UHRF1, a key factor for DNA methylation maintenance. Increasing Pramel7 expression in serum-cultured ESCs promotes a preimplantation epiblast-like gene signature, reduces UHRF1 levels and causes global DNA hypomethylation. Pramel7 is required for blastocyst formation and its forced expression locks ESCs in pluripotency. Pramel7/UHRF1 expression is mutually exclusive in ICMs whereas Pramel7-knockout embryos express high levels of UHRF1. Our data reveal an as-yet-unappreciated dynamic nature of DNA methylation through proteasome pathways and offer insights that might help to improve ESC culture to reproduce in vitro the in vivo ground-state pluripotency.

Identification of Novel Equine (Equus caballus) Tendon Markers Using RNA Sequencing.

Although several tendon-selective genes exist, they are also expressed in other musculoskeletal tissues. As cell and tissue engineering is reliant on specific molecular markers to discriminate between cell types, tendon-specific genes need to be identified. In order to accomplish this, we have used RNA sequencing (RNA-seq) to compare gene expression between tendon, bone, cartilage and ligament from horses. We identified several tendon-selective gene markers, and established eyes absent homolog 2 (EYA2) and a G-protein regulated inducer of neurite outgrowth 3 (GPRIN3) as specific tendon markers using RT-qPCR. Equine tendon cells cultured as three-dimensional spheroids expressed significantly greater levels of EYA2 than GPRIN3, and stained positively for EYA2 using immunohistochemistry. EYA2 was also found in fibroblast-like cells within the tendon tissue matrix and in cells localized to the vascular endothelium. In summary, we have identified EYA2 and GPRIN3 as specific molecular markers of equine tendon as compared to bone, cartilage and ligament, and provide evidence for the use of EYA2 as an additional marker for tendon cells in vitro.

lncRNA maturation to initiate heterochromatin formation in the nucleolus is required for exit from pluripotency in ESCs.

The open chromatin of embryonic stem cells (ESCs) condenses into repressive heterochromatin as cells exit the pluripotent state. How the 3D genome organization is orchestrated and implicated in pluripotency and lineage specification is not understood. Here, we find that maturation of the long noncoding RNA (lncRNA) pRNA is required for establishment of heterochromatin at ribosomal RNA genes, the genetic component of nucleoli, and this process is inactivated in pluripotent ESCs. By using mature pRNA to tether heterochromatin at nucleoli of ESCs, we find that localized heterochromatin condensation of ribosomal RNA genes initiates establishment of highly condensed chromatin structures outside of the nucleolus. Moreover, we reveal that formation of such highly condensed, transcriptionally repressed heterochromatin promotes transcriptional activation of differentiation genes and loss of pluripotency. Our findings unravel the nucleolus as an active regulator of chromatin plasticity and pluripotency and challenge current views on heterochromatin regulation and function in ESCs.