LINE-1 RNA triggers matrix formation in bone cells via a PKR-mediated inflammatory response.

Transposable elements (TEs) are mobile genetic modules of viral derivation that have been co-opted to become modulators of mammalian gene expression. TEs are a major source of endogenous dsRNAs, signaling molecules able to coordinate inflammatory responses in various physiological processes. Here, we provide evidence for a positive involvement of TEs in inflammation-driven bone repair and mineralization. In newly fractured mice bone, we observed an early transient upregulation of repeats occurring concurrently with the initiation of the inflammatory stage. In human bone biopsies, analysis revealed a significant correlation between repeats expression, mechanical stress and bone mineral density. We investigated a potential link between LINE-1 (L1) expression and bone mineralization by delivering a synthetic L1 RNA to osteoporotic patient-derived mesenchymal stem cells and observed a dsRNA-triggered protein kinase (PKR)-mediated stress response that led to strongly increased mineralization. This response was associated with a strong and transient inflammation, accompanied by a global translation attenuation induced by eIF2α phosphorylation. We demonstrated that L1 transfection reshaped the secretory profile of osteoblasts, triggering a paracrine activity that stimulated the mineralization of recipient cells.

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.

SAXS imaging reveals optimized osseointegration properties of bioengineered oriented 3D-PLGA/aCaP scaffolds in a critical size bone defect model.

Healing large bone defects remains challenging in orthopedic surgery and is often associated with poor outcomes and complications. A major issue with bioengineered constructs is achieving a continuous interface between host bone and graft to enhance biological processes and mechanical stability. In this study, we have developed a new bioengineering strategy to produce oriented biocompatible 3D PLGA/aCaP nanocomposites with enhanced osseointegration. Decellularized scaffolds -containing only extracellular matrix- or scaffolds seeded with adipose-derived mesenchymal stromal cells were tested in a mouse model for critical size bone defects. In parallel to micro-CT analysis, SAXS tensor tomography and 2D scanning SAXS were employed to determine the 3D arrangement and nanostructure within the critical-sized bone. Both newly developed scaffold types, seeded with cells or decellularized, showed high osseointegration, higher bone quality, increased alignment of collagen fibers and optimal alignment and size of hydroxyapatite minerals.

Polypyrimidine tract binding protein 1 regulates the activation of mouse CD8 T cells.

The RNA-binding protein polypyrimidine tract binding protein 1 (PTBP1) has been found to have roles in CD4 T-cell activation, but its function in CD8 T cells remains untested. We show it is dispensable for the development of naïve mouse CD8 T cells, but is necessary for the optimal expansion and production of effector molecules by antigen-specific CD8 T cells in vivo. PTBP1 has an essential role in regulating the early events following activation of the naïve CD8 T cell leading to IL-2 and TNF production. It is also required to protect activated CD8 T cells from apoptosis. PTBP1 controls alternative splicing of over 400 genes in naïve CD8 T cells in addition to regulating the abundance of ∼200 mRNAs. PTBP1 is required for the nuclear accumulation of c-Fos, NFATc2, and NFATc3, but not NFATc1. This selective effect on NFAT proteins correlates with PTBP1-promoted expression of the shorter Aß1 isoform and exon 13 skipped Aß2 isoform of the catalytic A-subunit of calcineurin phosphatase. These findings reveal a crucial role for PTBP1 in regulating CD8 T-cell activation.

High Frequency of Tumor Propagating Cells in Fusion-Positive Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Fusion-positive RMS (FPRMS), expressing the PAX3/7-FOXO1, has a worse prognosis compared to the more common fusion-negative RMS (FNRMS). Although several studies reported hierarchical organization for FNRMS with the identification of cancer stem cells, the cellular organization of FPRMS is not yet clear. In this study we investigated the expression of key stem cell markers, developed a sphere assay, and investigated the seven most common FPRMS cell lines for subpopulations of tumor propagating cancer stem-like cells, also called cancer stem cells (CSCs). Moreover, loss- and gain-of-functions of the stem cell genes SOX2, OCT4, and NANOG were investigated in the same cells. Single-cell clonal analysis was performed in vitro as well as in vivo. We found that no stable CSC subpopulation could be enriched in FPRMS. Unlike depletion of PAX3-FOXO1, neither overexpression nor siRNA-mediated downregulation of SOX2, OCT4, and NANOG affected physiology of RMS cells. Every single subclone-derived cell clone initiated tumor growth in mice, despite displaying considerable heterogeneity in gene expression. FPRMS appears to contain a high frequency of tumor propagating stem-like cells, which could explain their higher propensity for metastasis and relapse. Their dependency on PAX3-FOXO1 activity reinforces the importance of the fusion protein as the key therapeutic target.

Essential requirement for polypyrimidine tract binding proteins 1 and 3 in the maturation and maintenance of mature B cells in mice.

The maturation of immature B cells and the survival of mature B cells is stringently controlled to maintain a diverse repertoire of antibody specificities while avoiding self-reactivity. At the molecular level this is regulated by signaling from membrane Ig and the BAFF-receptor that sustain a pro-survival program of gene expression. Whether and how posttranscriptional mechanisms contribute to B cell maturation and survival remains poorly understood. Here, we show that the polypyrimidine tract binding proteins (PTBP) PTBP1 and PTBP3 bind to a large and overlapping set of transcripts in B cells. Both PTBP1 and PTBP3 bind to introns and exons where they are predicted to regulate alternative splicing. Moreover, they also show high-density of binding to 3' untranslated regions suggesting they influence the transcriptome in diverse ways. We show that PTBP1 and PTBP3 are required in B cells beyond the immature cell stage to sustain transitional B cells and the B1, marginal zone and follicular B cell lineages. Therefore, PTBP1 and PTBP3 promote the maturation of quiescent B cells by regulating gene expression at the posttranscriptional level.

Identification of ALP+/CD73+ defining markers for enhanced osteogenic potential in human adipose-derived mesenchymal stromal cells by mass cytometry.

BACKGROUND: The impressive progress in the field of stem cell research in the past decades has provided the ground for the development of cell-based therapy. Mesenchymal stromal cells obtained from adipose tissue (AD-MSCs) represent a viable source for the development of cell-based therapies. However, the heterogeneity and variable differentiation ability of AD-MSCs depend on the cellular composition and represent a strong limitation for their use in therapeutic applications. In order to fully understand the cellular composition of MSC preparations, it would be essential to analyze AD-MSCs at single-cell level. METHOD: Recent advances in single-cell technologies have opened the way for high-dimensional, high-throughput, and high-resolution measurements of biological systems. We made use of the cytometry by time-of-flight (CyTOF) technology to explore the cellular composition of 17 human AD-MSCs, interrogating 31 markers at single-cell level. Subcellular composition of the AD-MSCs was investigated in their naïve state as well as during osteogenic commitment, via unsupervised dimensionality reduction as well as supervised representation learning approaches. RESULT: This study showed a high heterogeneity and variability in the subcellular composition of AD-MSCs upon isolation and prolonged culture. Algorithm-guided identification of emerging subpopulations during osteogenic differentiation of AD-MSCs allowed the identification of an ALP+/CD73+ subpopulation of cells with enhanced osteogenic differentiation potential. We could demonstrate in vitro that the sorted ALP+/CD73+ subpopulation exhibited enhanced osteogenic potential and is moreover fundamental for osteogenic lineage commitment. We finally showed that this subpopulation was present in freshly isolated human adipose-derived stromal vascular fractions (SVFs) and that could ultimately be used for cell therapies. CONCLUSION: The data obtained reveal, at single-cell level, the heterogeneity of AD-MSCs from several donors and highlight how cellular composition impacts the osteogenic differentiation capacity. The marker combination (ALP/CD73) can not only be used to assess the differentiation potential of undifferentiated AD-MSC preparations, but also could be employed to prospectively enrich AD-MSCs from the stromal vascular fraction of human adipose tissue for therapeutic applications.

Polypyrimidine tract-binding proteins are essential for B cell development.

Polypyrimidine tract-binding protein 1 (PTBP1) is a RNA-binding protein (RBP) expressed throughout B cell development. Deletion of Ptbp1 in mouse pro-B cells results in upregulation of PTBP2 and normal B cell development. We show that PTBP2 compensates for PTBP1 in B cell ontogeny as deletion of both Ptbp1 and Ptbp2 results in a complete block at the pro-B cell stage and a lack of mature B cells. In pro-B cells PTBP1 ensures precise synchronisation of the activity of cyclin dependent kinases at distinct stages of the cell cycle, suppresses S-phase entry and promotes progression into mitosis. PTBP1 controls mRNA abundance and alternative splicing of important cell cycle regulators including CYCLIN-D2, c-MYC, p107 and CDC25B. Our results reveal a previously unrecognised mechanism mediated by a RBP that is essential for B cell ontogeny and integrates transcriptional and post-translational determinants of progression through the cell cycle.

Autologous endothelialized small-caliber vascular grafts engineered from blood-derived induced pluripotent stem cells.

An ideal cell source for human therapeutic and disease modeling applications should be easily accessible and possess unlimited differentiation and expansion potential. Human induced pluripotent stem cells (hiPSCs) derived from peripheral blood mononuclear cells (PBMCs) represent a promising source given their ease of harvest and their pluripotent nature. Previous studies have demonstrated the feasibility of using PBMC-derived hiPSCs for vascular tissue engineering. However, so far, no endothelialization of hiPSC-derived tissue engineered vascular grafts (TEVGs) based on fully biodegradable polymers without xenogenic matrix components has been shown. In this study, we have generated hiPSCs from PBMCs and differentiated them into αSMA- and calponin-positive smooth muscle cells (SMCs) as well as endothelial cells (ECs) positive for CD31, vWF and eNOS. Both cell types were co-seeded on PGA-P4HB starter matrices and cultured under static or dynamic conditions to induce tissue formation in vitro. The resulting small diameter vascular grafts showed abundant amounts of extracellular matrix, containing a thin luminal layer of vWF-positive cells and a subendothelial αSMA-positive layer approximating the architecture of native vessels. Our results demonstrate the successful generation of TEVGs based on SMCs and ECs differentiated from PBMC-derived hiPSC combined with a biodegradable polymer. These results pave the way for developing autologous PBMC-derived hiPSC-based vascular constructs for therapeutic applications or disease modeling. STATEMENT OF SIGNIFICANCE: We report for the first time the possibility to employ human peripheral blood mononuclear cell (PBMC)-derived iPSCs to generate biodegradable polymer-based tissue engineered vascular grafts (TEVG), which mimic the native layered architecture of blood vessels. hiPSCs from PBMCs were differentiated into smooth muscle cells as well as endothelial cells. These cells were co-seeded on a biodegradable PGA/P4HB scaffold and cultured in a bioreactor to induce tissue formation in vitro. The resulting small diameter TEVG showed abundant amounts of extracellular matrix, containing a αSMA-positive layer in the interstitium and a thin luminal layer of vWF-positive endothelial cells approximating the architecture of native vessels. Our findings improving the generation of autologous vascular replacements using blood as an easily accessible cell source.

Automated digital image quantification of histological staining for the analysis of the trilineage differentiation potential of mesenchymal stem cells.

BACKGROUND: Multipotent mesenchymal stem cells (MSCs) have the potential to repair and regenerate damaged tissues and are considered as attractive candidates for the development of cell-based regenerative therapies. Currently, there are more than 200 clinical trials involving the use of MSCs for a wide variety of indications. However, variations in their isolation, expansion, and particularly characterization have made the interpretation of study outcomes or the rigorous assessment of therapeutic efficacy difficult. An unbiased characterization of MSCs is of major importance and essential to guaranty that only the most suitable cells will be used. The development of standardized and reproducible assays to predict MSC potency is therefore mandatory. The currently used quantification methodologies for the determination of the trilineage potential of MSCs are usually based on absorbance measurements which are imprecise and prone to errors. We therefore aimed at developing a methodology first offering a standardized way to objectively quantify the trilineage potential of MSC preparations and second allowing to discriminate functional differences between clonally expanded cell populations. METHOD: MSCs originating from several patients were differentiated into osteoblasts, adipocytes, and chondroblasts for 14, 17, and 21 days. Differentiated cells were then stained with the classical dyes: Alizarin Red S for osteoblasts, Oil Red O for adipocytes, and Alcian Blue 8GX for chondroblasts. Quantification of differentiation was then performed with our newly developed digital image analysis (DIA) tool followed by the classical absorbance measurement. The results from the two techniques were then compared. RESULT: Quantification based on DIA allowed highly standardized and objective dye quantification with superior sensitivity compared to absorbance measurements. Furthermore, small differences between MSC lines in the differentiation potential were highlighted using DIA whereas no difference was detected using absorbance quantification. CONCLUSION: Our approach represents a novel method that simplifies the laboratory procedures not only for the quantification of histological dyes and the degree of differentiation of MSCs, but also due to its color independence, it can be easily adapted for the quantification of a wide range of staining procedures in histology. The method is easily applicable since it is based on open source software and standard light microscopy.

Transcriptome Analysis of Infected and Bystander Type 2 Alveolar Epithelial Cells during Influenza A Virus Infection Reveals In Vivo Wnt Pathway Downregulation.

Influenza virus outbreaks remain a serious threat to public health. A greater understanding of how cells targeted by the virus respond to the infection can provide insight into the pathogenesis of disease. Here we examined the transcriptional profile of in vivo-infected and uninfected type 2 alveolar epithelial cells (AEC) in the lungs of influenza virus-infected mice. We show for the first time the unique gene expression profiles induced by the in vivo infection of AEC as well as the transcriptional response of uninfected bystander cells. This work allows us to distinguish the direct and indirect effects of infection at the cellular level. Transcriptome analysis revealed that although directly infected and bystander AEC from infected animals shared many transcriptome changes compared to AEC from uninfected animals, directly infected cells produce more interferon and express lower levels of Wnt signaling-associated transcripts, while concurrently expressing more transcripts associated with cell death pathways, than bystander uninfected AEC. The Wnt signaling pathway was downregulated in both in vivo-infected AEC and in vitro-infected human lung epithelial A549 cells. Wnt signaling did not affect type I and III interferon production by infected A549 cells. Our results reveal unique transcriptional changes that occur within infected AEC and show that influenza virus downregulates Wnt signaling. In light of recent findings that Wnt signaling is essential for lung epithelial stem cells, our findings reveal a mechanism by which influenza virus may affect host lung repair.IMPORTANCE Influenza virus infection remains a major public health problem. Utilizing a recombinant green fluorescent protein-expressing influenza virus, we compared the in vivo transcriptomes of directly infected and uninfected bystander cells from infected mouse lungs and discovered many pathways uniquely regulated in each population. The Wnt signaling pathway was downregulated in directly infected cells and was shown to affect virus but not interferon production. Our study is the first to discern the in vivo transcriptome changes induced by direct viral infection compared to mere exposure to the lung inflammatory milieu and highlight the downregulation of Wnt signaling. This downregulation has important implications for understanding influenza virus pathogenesis, as Wnt signaling is critical for lung epithelial stem cells and lung epithelial cell differentiation. Our findings reveal a mechanism by which influenza virus may affect host lung repair and suggest interventions that prevent damage or accelerate recovery of the lung.

The RNA-binding protein PTBP1 is necessary for B cell selection in germinal centers.

Antibody affinity maturation occurs in germinal centers (GCs), where B cells cycle between the light zone (LZ) and the dark zone. In the LZ, GC B cells bearing immunoglobulins with the highest affinity for antigen receive positive selection signals from helper T cells, which promotes their rapid proliferation. Here we found that the RNA-binding protein PTBP1 was needed for the progression of GC B cells through late S phase of the cell cycle and for affinity maturation. PTBP1 was required for proper expression of the c-MYC-dependent gene program induced in GC B cells receiving T cell help and directly regulated the alternative splicing and abundance of transcripts that are increased during positive selection to promote proliferation.

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.

Characterization of the B Cell Transcriptome Bound by RNA-Binding Proteins with iCLIP.

Posttranscriptional regulation of gene expression shapes the B cell transcriptome and controls messenger RNA (mRNA) translation into protein. Recent reports have highlighted the importance of RNA binding proteins (RBPs) for mRNA splicing, subcellular location, stability, and translation during B lymphocyte development, activation, and differentiation. Here we describe individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) in primary lymphocytes, a method that maps RNA-protein interactions in a genome-wide scale allowing mechanistic analysis of RBP function. We discuss the latest improvements in iCLIP technology and provide some examples of how integration of the RNA-protein interactome with other high-throughput mRNA sequencing methodologies uncovers the important role of RBP-mediated RNA regulation in key biological cell processes.

RNA-binding proteins ZFP36L1 and ZFP36L2 promote cell quiescence.

Progression through the stages of lymphocyte development requires coordination of the cell cycle. Such coordination ensures genomic integrity while cells somatically rearrange their antigen receptor genes [in a process called variable-diversity-joining (VDJ) recombination] and, upon successful rearrangement, expands the pools of progenitor lymphocytes. Here we show that in developing B lymphocytes, the RNA-binding proteins (RBPs) ZFP36L1 and ZFP36L2 are critical for maintaining quiescence before precursor B cell receptor (pre-BCR) expression and for reestablishing quiescence after pre-BCR-induced expansion. These RBPs suppress an evolutionarily conserved posttranscriptional regulon consisting of messenger RNAs whose protein products cooperatively promote transition into the S phase of the cell cycle. This mechanism promotes VDJ recombination and effective selection of cells expressing immunoglobulin-µ at the pre-BCR checkpoint.

The Forgotten: Identification and Functional Characterization of MHC Class II Molecules H2-Eb2 and RT1-Db2.

In this article, we report the complete coding sequence and to our knowledge, the first functional analysis of two homologous nonclassical MHC class II genes: RT1-Db2 of rat and H2-Eb2 of mouse. They differ in important aspects compared with the classical class II ß1 molecules: their mRNA expression by APCs is much lower, they show minimal polymorphism in the Ag-binding domain, and they lack N-glycosylation and the highly conserved histidine 81. Also, their cytoplasmic region is completely different and longer. To study and compare them with their classical counterparts, we transduced them in different cell lines. These studies show that they can pair with the classical α-chains (RT1-Da and H2-Ea) and are expressed at the cell surface where they can present superantigens. Interestingly, compared with the classical molecules, they have an extraordinary capacity to present the superantigen Yersinia pseudotuberculosis mitogen. Taken together, our findings suggest that the b2 genes, together with the respective α-chain genes, encode for H2-E2 or RT1-D2 molecules, which could function as Ag-presenting molecules for a particular class of Ags, as modulators of Ag presentation like nonclassical nonpolymorphic class II molecules DM and DO do, or even as players outside the immune system.

Direct transplantation of native pericytes from adipose tissue: A new perspective to stimulate healing in critical size bone defects.

BACKGROUND AIMS: Fractures with a critical size bone defect (e.g., open fracture with segmental bone loss) are associated with high rates of delayed union and non-union. The prevention and treatment of these complications remain a serious issue in trauma and orthopaedic surgery. Autologous cancellous bone grafting is a well-established and widely used technique. However, it has drawbacks related to availability, increased morbidity and insufficient efficacy. Mesenchymal stromal cells can potentially be used to improve fracture healing. In particular, human fat tissue has been identified as a good source of multilineage adipose-derived stem cells, which can be differentiated into osteoblasts. The main issue is that mesenchymal stromal cells are a heterogeneous population of progenitors and lineage-committed cells harboring a broad range of regenerative properties. This heterogeneity is also mirrored in the differentiation potential of these cells. In the present study, we sought to test the possibility to enrich defined subpopulations of stem/progenitor cells for direct therapeutic application without requiring an in vitro expansion. METHODS: We enriched a CD146+NG2+CD45- population of pericytes from freshly isolated stromal vascular fraction from mouse fat tissue and tested their osteogenic differentiation capacity in vitro and in vivo in a mouse model for critical size bone injury. RESULTS: Our results confirm the ability of enriched CD146+NG2+CD45- cells to efficiently generate osteoblasts in vitro, to colonize cancellous bone scaffolds and to successfully contribute to regeneration of large bone defects in vivo. CONCLUSIONS: This study represents proof of principle for the direct use of enriched populations of cells with stem/progenitor identity for therapeutic applications.

Generation of functionally distinct isoforms of PTBP3 by alternative splicing and translation initiation.

Polypyrimidine tract binding protein (PTBP1) is a widely expressed RNA binding protein that acts as a regulator of alternative splicing and of cytoplasmic mRNA functions. Vertebrates contain two closely-related paralogs with >75% amino acid sequence identity. Early replacement of PTBP1 by PTBP2 during neuronal differentiation causes a concerted set of splicing changes. By comparison, very little is known about the molecular functions or physiological roles of PTBP3, although its expression and conservation throughout the vertebrates suggest a role in haematopoietic cells. To begin to understand its functions we have characterized the mRNA and protein isoform repertoire of PTBP3. Combinatorial alternative splicing events at the 5' end of the gene allow for the generation of eight mRNA and three major protein isoforms. Individual mRNAs generate up to three protein isoforms via alternative translation initiation by re-initiation and leaky scanning using downstream AUG codons. The N-terminally truncated PTBP3 isoforms lack nuclear localization signals and/or most of the RRM1 domain and vary in their RNA binding properties and nuclear/cytoplasmic distribution, suggesting that PTBP3 may have major post-transcriptional cytoplasmic roles. Our findings set the stage for understanding the non-redundant physiological roles of PTBP3.