Serum concentration of dickkopf-related protein 1 (DKK1) in psoriatic arthritis in the context of bone remodelling.

Psoriatic arthritis (PsA) is a chronic inflammatory disease, characterised by the pathological occurrence of two opposite phenomena-osteoresorption and osteogenesis. Dickkopf-related protein 1 (DKK1) which inhibits the Wingless protein (Wnt) signalling pathway has been shown to be a master regulator of bone remodeling in inflammatory rheumatic diseases. However, the exact relationship between DKK1 serum level and bone remodelling is not clear. The goal of this study is to review state-of-the-art knowledge on the association of serum DKK1 with a bone remodelling in PsA. The MEDLINE-PubMed, EMBASE, Scopus, Web of Science and DOAJ databases were searched for appropriate papers. The English terms: 'DKK1', 'Dickkopf-1' 'Dickkopf related protein 1', 'psoriatic arthritis' and 'PsA' were used for search purposes. Eight original articles and two reviews were identified up to August 2023. In four out of 8 discussed studies DKK1 serum level was higher in PsA patients than in healthy controls [Dalbeth, p < 0.01; Diani, p < 0.001; Chung, p < 0.01; Abd el Hamid, p < 0.001)], it was comparable in another (Daousiss, p = 0.430) and was lower in two (Fassio2017, p < 0.05; Fassio2019, p < 0.05). In one study, the comparative groups included patients with axial spondyloarthritis, where DKK1 serum levels were lower in PsA groups [Jadon, peripheral PsA, p = 0.01]. The true relative serum concentration of DKK1 in PsA, as well as its influence on osteogenesis and osteoresorption, is still equivocal. Further studies on this matter with consistent and stringent methodology are warranted.

WNT/ß-Catenin Signaling Promotes TGF-ß-Mediated Activation of Human Cardiac Fibroblasts by Enhancing IL-11 Production.

Cardiac fibrosis is a pathological process associated with the development of heart failure. TGF-ß and WNT signaling have been implicated in pathogenesis of cardiac fibrosis, however, little is known about molecular cross-talk between these two pathways. The aim of this study was to examine the effect of exogenous canonical WNT3a and non-canonical WNT5a in TGF-ß-activated human cardiac fibroblasts. We found that WNT3a and TGF-ß induced a ß-catenin-dependent response, whereas WNT5a prompted AP-1 activity. TGF-ß triggered profibrotic signatures in cardiac fibroblasts, and co-stimulation with WNT3a or co-activation of the ß-catenin pathway with the GSK3ß inhibitor CHIR99021 enhanced collagen I and fibronectin production and development of active contractile stress fibers. In the absence of TGF-ß, neither WNT3a nor CHIR99021 exerted profibrotic responses. On a molecular level, in TGF-ß-activated fibroblasts, WNT3a enhanced phosphorylation of TAK1 and production and secretion of IL-11 but showed no effect on the Smad pathway. Neutralization of IL-11 activity with the blocking anti-IL-11 antibody effectively reduced the profibrotic response of cardiac fibroblasts activated with TGF-ß and WNT3a. In contrast to canonical WNT3a, co-activation with non-canonical WNT5a suppressed TGF-ß-induced production of collagen I. In conclusion, WNT/ß-catenin signaling promotes TGF-ß-mediated fibroblast-to-myofibroblast transition by enhancing IL-11 production. Thus, the uncovered mechanism broadens our knowledge on a molecular basis of cardiac fibrogenesis and defines novel therapeutic targets for fibrotic heart diseases.

Heart non-specific effector CD4+ T cells protect from postinflammatory fibrosis and cardiac dysfunction in experimental autoimmune myocarditis.

Heart-specific CD4+ T cells have been implicated in development and progression of myocarditis in mice and in humans. Here, using mouse models of experimental autoimmune myocarditis (EAM) we investigated the role of heart non-specific CD4+ T cells in the progression of the disease. Heart non-specific CD4+ T cells were obtained from DO11.10 mice expressing transgenic T cell receptor recognizing chicken ovalbumin. We found that heart infiltrating CD4+ T cells expressed exclusively effector (Teff) phenotype in the EAM model and in hearts of patients with lymphocytic myocarditis. Adoptive transfer experiments showed that while heart-specific Teff infiltrated the heart shortly after injection, heart non-specific Teff effectively accumulated during myocarditis and became the major heart-infiltrating CD4+ T cell subset at later stage. Restimulation of co-cultured heart-specific and heart non-specific CD4+ T cells with alpha-myosin heavy chain antigen showed mainly Th1/Th17 response for heart-specific Teff and up-regulation of a distinct set of extracellular signalling molecules in heart non-specific Teff. Adoptive transfer of heart non-specific Teff in mice with myocarditis did not affect inflammation severity at the peak of disease, but protected the heart from adverse post-inflammatory fibrotic remodelling and cardiac dysfunction at later stages of disease. Furthermore, mouse and human Teff stimulated in vitro with common gamma cytokines suppressed expression of profibrotic genes, reduced amount of α-smooth muscle actin filaments and decreased contraction of cardiac fibroblasts. In this study, we provided a proof-of-concept that heart non-specific Teff cells could effectively contribute to myocarditis and protect the heart from the dilated cardiomyopathy outcome.

WNT3a and WNT5a Transported by Exosomes Activate WNT Signaling Pathways in Human Cardiac Fibroblasts.

WNT signaling plays an important role in fibrotic processes in the heart. Recently, exosomes have been proposed as novel extracellular transporters for WNT proteins. In this study, we analyzed whether WNT3a and WNT5a carried by exosomes could activate downstream molecular pathways in human cardiac fibroblasts. Exosomes were isolated from conditioned medium of control, WNT3a- and WNT5a-producing L cells by differential ultracentrifugations. Obtained exosomes showed size ranging between 20⁻150 nm and expressed exosomal markers ALG-2-interacting protein X (ALIX) and CD63. Treatment with WNT3a-rich exosomes inhibited activity of glycogen synthase kinase 3ß (GSK3ß), induced nuclear translocation of ß-catenin, and activated T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factors as well as expression of WNT/ß-catenin responsive genes in cardiac fibroblasts, but did not coactivate extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and activator protein 1 (AP-1) signaling pathways. In contrast, exosomes produced by WNT5a-producing L cells failed to activate ß-catenin-dependent response, but successfully triggered phosphorylation of ERK1/2 and JNK and stimulated IL-6 production. In conclusion, exosomes containing WNT proteins can functionally contribute to cardiac fibrosis by activating profibrotic WNT pathways on cardiac fibroblasts and may represent a novel mechanism of spreading profibrotic signals in the heart.

Sera of patients with axial spondyloarthritis (axSpA) enhance osteoclastogenic potential of monocytes isolated from healthy individuals.

BACKGROUND: Axial spondyloarthritis (axSpA) is characterized by significant bone loss caused by dysregulation of physiological bone turnover, possibly resulting from intensified differentiation of osteoclasts. The aim of this study was to reevaluate the levels of osteoclastogenesis-mediating factors: soluble RANKL, M-CSF, OPG and other cytokines in sera of untreated, with sDMARDs and/or bDMARDs, axSpA patients and to test whether these sera influence differentiation of healthy monocytes towards osteoclast lineage. METHODS: Bone remodeling molecules (RANKL, M-CSF, OPG, IL-6, OSM, IL-17A, TGFß, and TNFα) were evaluated in 27 patients with axSpA and 23 age and sex-matched controls. Disease activity (BASDAI, ASDAS) and inflammatory markers (ESR, CRP) were assessed. Monocytes obtained from healthy individuals were cultured in vitro in presence of sera from 11 randomly chosen axSpA patients and 10 controls, with addition of exogenous M-CSF and/or RANKL or without. Osteoclastic differentiation was assessed analyzing osteoclast markers (cathepsin K and RANK at mRNA level) and with osteoclast-specific staining. RESULTS: axSpA patients' sera levels of soluble RANKL were significantly lower and M-CSF, IL-6, OSM, IL-17A and TNFα significantly higher in comparison to controls, whereas of OPG and TGFß were comparable in both groups. Numbers of generated in vitro osteoclasts and cathepsin K mRNA levels did not differ between cultures supplemented with sera of healthy and axSpA patients, both in the absence and presence of M-CSF. Instead, addition of exogenous RANKL boosted osteoclastogenesis, which was significantly higher in cultures with axSpA sera. Furthermore, sera from axSpA patients induced substantially higher levels of RANK mRNA, independently of M-CSF and RANKL stimulation. CONCLUSION: We show that, paradoxically, serum levels of soluble RANKL observed in axSpA are in fact significantly lower in comparison to healthy blood donors. Our results indicate that sera of axSpA patients - in contrary to healthy subjects - contain circulating, soluble factors (presumably IL-6, OSM, IL-17A, TNFα and others) able to stimulate healthy monocytes responsiveness to even relative low RANKL serum levels, by inducing high RANK mRNA expression and - as a net effect - boosting their osteoclastogenic potential. We suggest also that locally produced RANKL in axSpA may induce overactive osteoclasts from their precursors.

Characterization and comparison of osteoblasts derived from mouse embryonic stem cells and induced pluripotent stem cells.

New developments in stem cell biology offer alternatives for the reconstruction of critical-sized bone defects. One of these developments is the use of induced pluripotent stem (iPS) cells. These stem cells are similar to embryonic stem (ES) cells, but can be generated from adult somatic cells and therefore do not raise ethical concerns. Proper characterization of iPS-derived osteoblasts is important for future development of safe clinical applications of these cells. For this reason, we differentiated mouse ES and iPS cells toward osteoblasts using osteogenic medium and compared their functionality. Immunocytochemical analysis showed significant expression of bone markers (osteocalcin and collagen type I) in osteoblasts differentiated from ES and iPS cells on days 7 and 30. An in vitro mineralization assay confirmed the functionality of osteogenically differentiated ES and iPS cells. Gene expression arrays focusing on osteogenic differentiation were performed in order to compare the gene expression pattern in both differentiated and undifferentiated ES cells and iPS cells. We observed a significant upregulation of osteogenesis-related genes such as Runx2, osteopontin, collagen type I, Tnfsf11, Csf1, and alkaline phosphatase upon osteogenic differentiation of the ES and iPS cells. We further validated the expression of key osteogenic genes Runx2, osteopontin, osteocalcin, collagen type I, and osterix in both differentiated and undifferentiated ES and iPS cells by means of quantified real-time polymerase chain reaction. We conclude that ES and iPS cells are similar in their osteogenic differentiation capacities, as well as in their gene expression patterns.

Human oligodendrocytes in remyelination research.

Studies on myelination and oligodendrocyte development are inevitably linked with demyelinating conditions such as multiple sclerosis (MS), leukodystrophies or spinal cord injury (SCI). Chronic loss of myelin, subsequently leading to neurodegeneration, is the ultimate cause of severe and permanent disability. Thus, fast restoration of myelin (remyelination) is essential for circumventing demyelination-caused pathologies. Implantation of exogenous remyelinating cells has been considered as a potential remyelination strategy. Researchers have examined a variety of cell types endowed with myelin-forming capacity (oligodendrocytes, Schwann cells, olfactory ensheathing cells etc.) in vitro and in vivo for their potential application as myelin restoring cell grafts. This review gives a summary of studies on the generation and testing of pure suspensions of human oligodendrocytes as a clinically relevant, efficient cellular tool for treating myelin pathology. We start with a brief overview of the current knowledge on the development of human oligodendrocytes from the late stages of embryogenesis up to the early postnatal stage. Insight in the specific extrinsic and intrinsic factors regulating normal oligodendrogenesis is crucial in order to achieve and maintain a sufficient population of engraftable functional oligodendrocytes in vitro. We discuss potential sources of human oligodendrocytes, including novel oligodendrocyte generation strategies employing induced pluripotent stem cells (iPSCs) and direct conversion technology. Finally, we provide a systematic overview of (the outcome of) experimental studies, in which human oligodendrocytes were tested for their (re)myelination capacity and efficiency.

Monocyte subpopulations display disease-specific miRNA signatures depending on the subform of Spondyloarthropathy.

Spondyloarthropathies (SpA) are a family of rheumatic disorders that could be divided into axial (axSpA) and peripheral (perSpA) sub-forms depending on the disease clinical presentation. The chronic inflammation is believed to be driven by innate immune cells such as monocytes, rather than self-reactive cells of adaptive immune system. The aim of the study was to investigate the micro-RNA (miRNA) profiles in monocyte subpopulations (classical, intermediate and non-classical subpopulations) acquired from SpA patients or healthy individuals in search for prospective disease specific and/or disease subtype differentiating miRNA markers. Several SpA-specific and axSpA/perSpA differentiating miRNAs have been identified that appear to be characteristic for specific monocyte subpopulation. For classical monocytes, upregulation of miR-567 and miR-943 was found to be SpA-specific, whereas downregulation of miR-1262 could serve as axSpA-differentiating, and the expression pattern of miR-23a, miR-34c, mi-591 and miR-630 as perSpA-differentiating markers. For intermediate monocytes, expression levels of miR-103, miR-125b, miR-140, miR-374, miR-376c and miR-1249 could be used to distinguish SpA patients from healthy donors, whereas the expression pattern of miR-155 was identified as characteristic for perSpA. For non-classical monocytes, differential expression of miR-195 was recognized as general SpA indicator, while upregulation of miR-454 and miR-487b could serve as axSpA-differentiating, and miR-1291 as perSpA-differentiating markers. Our data indicate for the first time that in different SpA subtypes, monocyte subpopulations bear disease-specific miRNA signatures that could be relevant for SpA diagnosis/differentiation process and may help to understand SpA etiopathology in the context of already known functions of monocyte subpopulations.

T Lymphocyte-Derived Exosomes Transport MEK1/2 and ERK1/2 and Induce NOX4-Dependent Oxidative Stress in Cardiac Microvascular Endothelial Cells.

Background: Activation of endothelial cells by inflammatory mediators secreted by CD4+ T lymphocytes plays a key role in the inflammatory response. Exosomes represent a specific class of signaling cues transporting a mixture of proteins, nucleic acids, and other biomolecules. So far, the impact of exosomes shed by T lymphocytes on cardiac endothelial cells remained unknown. Methods and Results: Supernatants of CD4+ T cells activated with anti-CD3/CD28 beads were used to isolate exosomes by differential centrifugation. Activation of CD4+ T cells enhanced exosome production, and these exosomes (CD4-exosomes) induced oxidative stress in cardiac microvascular endothelial cells (cMVECs) without affecting their adhesive properties. Furthermore, CD4-exosome treatment aggravated the generation of mitochondrial reactive oxygen species (ROS), reduced nitric oxide (NO) levels, and enhanced the proliferation of cMVECs. These effects were reversed by adding the antioxidant apocynin. On the molecular level, CD4-exosomes increased NOX2, NOX4, ERK1/2, and MEK1/2 in cMVECs, and ERK1/2 and MEK1/2 proteins were found in CD4-exosomes. Inhibition of either MEK/ERK with U0126 or ERK with FR180204 successfully protected cMVECs from increased ROS levels and reduced NO bioavailability. Treatment with NOX1/4 inhibitor GKT136901 effectively blocked excessive ROS and superoxide production, reversed impaired NO levels, and reversed enhanced cMVEC proliferation triggered by CD4-exosomes. The siRNA-mediated silencing of Nox4 in cMVECs confirmed the key role of NOX4 in CD4-exosome-induced oxidative stress. To address the properties of exosomes under inflammatory conditions, we used the mouse model of CD4+ T cell-dependent experimental autoimmune myocarditis. In contrast to exosomes obtained from control hearts, exosomes obtained from inflamed hearts upregulated NOX2, NOX4, ERK1/2, MEK1/2, increased ROS and superoxide levels, and reduced NO bioavailability in treated cMVECs, and these changes were reversed by apocynin. Conclusion: Our results point to exosomes as a novel class of bioactive factors secreted by CD4+ T cells in immune response and represent potential important triggers of NOX4-dependent endothelial dysfunction. Neutralization of the prooxidative aspect of CD4-exosomes could open perspectives for the development of new therapeutic strategies in inflammatory cardiovascular diseases.

Angiotensin II receptor 1 controls profibrotic Wnt/ß-catenin signalling in experimental autoimmune myocarditis.

AIMS: Angiotensin (Ang) II signalling has been suggested to promote cardiac fibrosis in inflammatory heart diseases; however, the underlying mechanisms remain obscure. Using Agtr1a-/- mice with genetic deletion of angiotensin receptor type 1 (ATR1) and the experimental autoimmune myocarditis (EAM) model, we aimed to elucidate the role of Ang II-ATR1 pathway in development of heart-specific autoimmunity and post-inflammatory fibrosis. METHODS AND RESULTS: EAM was induced in wild-type (WT) and Agtr1a-/- mice by subcutaneous injections with alpha myosin heavy chain peptide emulsified in complete Freund's adjuvant. Agtr1a-/- mice developed myocarditis to a similar extent as WT controls at day 21 but showed reduced fibrosis and better systolic function at day 40. Crisscross bone marrow chimaera experiments proved that ATR1 signalling in the bone marrow compartment was critical for cardiac fibrosis. Heart infiltrating, bone-marrow-derived cells produced Ang II, but lack of ATR1 in these cells reduced transforming growth factor beta (TGF-ß)-mediated fibrotic responses. At the molecular level, Agtr1a-/- heart-inflammatory cells showed impaired TGF-ß-mediated phosphorylation of Smad2 and TAK1. In WT cells, TGF-ß induced formation of RhoA-GTP and RhoA-A-kinase anchoring protein-Lbc (AKAP-Lbc) complex. In Agtr1a-/- cells, stabilization of RhoA-GTP and interaction of RhoA with AKAP-Lbc were largely impaired. Furthermore, in contrast to WT cells, Agtr1a-/- cells stimulated with TGF-ß failed to activate canonical Wnt pathway indicated by suppressed activity of glycogen synthase kinase-3 (GSK-3)ß and nuclear ß-catenin translocation and showed reduced expression of Wnts. In line with these in vitro findings, ß-catenin was detected in inflammatory regions of hearts of WT, but not Agtr1a-/- mice and expression of canonical Wnt1 and Wnt10b were lower in Agtr1a-/- hearts. CONCLUSION: Ang II-ATR1 signalling is critical for development of post-inflammatory fibrotic remodelling and dilated cardiomyopathy. Our data underpin the importance of Ang II-ATR1 in effective TGF-ß downstream signalling response including activation of profibrotic Wnt/ß-catenin pathway.

Differentiation of induced pluripotent stem cells into functional oligodendrocytes.

The technology to generate autologous pluripotent stem cells (iPS cells) from almost any somatic cell type has brought various cell replacement therapies within clinical research. Besides the challenge to optimize iPS protocols to appropriate safety and GMP levels, procedures need to be developed to differentiate iPS cells into specific fully differentiated and functional cell types for implantation purposes. In this article, we describe a protocol to differentiate mouse iPS cells into oligodendrocytes with the aim to investigate the feasibility of IPS stem cell-based therapy for demyelinating disorders, such as multiple sclerosis. Our protocol results in the generation of oligodendrocyte precursor cells (OPCs) that can develop into mature, myelinating oligodendrocytes in-vitro (co-culture with DRG neurons) as well as in-vivo (after implantation in the demyelinated corpus callosum of cuprizone-treated mice). We report the importance of complete purification of the iPS-derived OPC suspension to prevent the contamination with teratoma-forming iPS cells.

Down-Regulation of Dkk-1 in Platelets of Patients With Axial Spondyloarthritis.

OBJECTIVE: Axial spondyloarthritis (SpA) is a chronic autoinflammatory disease with new bone formation, which is controlled by Wnt/ß-catenin signaling. Dkk-1 is an inhibitor of the Wnt pathway, and in humans, platelets represent a major source of Dkk-1. This study was undertaken to investigate whether levels of Dkk-1 in serum and platelet expression of DKK1 messenger RNA (mRNA) and Dkk-1 protein are affected in patients with axial SpA compared to healthy controls. METHODS: Forty-one patients with axial SpA and 35 healthy controls were enrolled in the study. Total serum Dkk-1 levels in all patients and healthy controls were measured by quantitative enzyme-linked immunosorbent assay. Platelet DKK1 mRNA was analyzed by quantitative reverse transcriptase-polymerase chain reaction in 20 patients with axial SpA and 20 controls, and Dkk-1 protein levels were measured by immunoblotting in 20 patients with axial SpA and 18 controls. RESULTS: We found a lower concentration of Dkk-1 in the serum from patients with axial SpA compared to the serum from healthy controls (P < 0.0001). Furthermore, the expression of Dkk-1 was significantly reduced both at the transcriptional level (P < 0.04) and at the protein level (P < 0.007) in platelets isolated from the blood of patients with axial SpA. CONCLUSION: Our preliminary observations suggest that dysfunction of the megakaryocyte/platelet axis might be responsible for reduced serum Dkk-1 levels in patients with axial SpA. Dkk-1 is down-regulated in the platelets of patients with axial SpA, a mechanism that might play a role in new bone formation.

Expression of VEGFA-mRNA in classical and MSX2-mRNA in non-classical monocytes in patients with spondyloarthritis is associated with peripheral arthritis.

Spondyloarthritis (SpA) is characterized by chronic inflammation and structural damage involving spine and peripheral joints. Monocytes, as part of innate immune system, following migration into affected tissue, may play a role in the pathogenesis of SpA. Here, potential associations between osteogenesis-linked gene expression profile in particular monocyte subpopulations and clinical signs of SpA were investigated. The 20 patients with axial and 16 with peripheral SpA were enrolled in the study. Monocyte subpopulations (classical-CD14++CD16-, intermediate-CD14++CD16+ and non-classical-CD14+CD16++) were isolated from blood using flow cytometry and gene expression analysis was performed using real-time PCR method and TaqMan Array, Human Osteogenesis, Fast 96-well plates. Next, the characteristic clinical features shared by axial and peripheral SpA were analyzed in the context of the expression of selected genes in the three subpopulations of monocytes. We demonstrated that expression of VEGFA in classical and MSX2 in non-classical monocytes were associated with the number of swollen and painful peripheral joints of SpA patients. We conclude that monocytes may contribute to the development of peripheral arthritis in SpA patients. This might be possible through subpopulation specific effects, linking number of inflamed joints with expression of VEGFA in classical monocytes and MSX2 in non-classical monocytes.

Human and mouse PD-L1: similar molecular structure, but different druggability profiles.

In the development of PD-L1-blocking therapeutics, it is essential to transfer initial in vitro findings into proper in vivo animal models. Classical immunocompetent mice are attractive due to high accessibility and low experimental costs. However, it is unknown whether inter-species differences in PD-L1 sequence and structure would allow for human-mouse cross applications. Here, we disclose the first structure of the mouse (m) PD-L1 and analyze its similarity to the human (h) PD-L1. We show that mPD-L1 interacts with hPD-1 and provides a negative signal toward activated Jurkat T cells. We also show major differences in druggability between the hPD-L1 and mPD-L1 using therapeutic antibodies, a macrocyclic peptide, and small molecules. Our study indicates that while the amino acid sequence is well conserved between the hPD-L1 and mPD-L1 and overall structures are almost identical, crucial differences determine the interaction with anti-PD-L1 agents, that cannot be easily predicted in silico.

Haploinsufficient Rock1+/- and Rock2+/- Mice Are Not Protected from Cardiac Inflammation and Postinflammatory Fibrosis in Experimental Autoimmune Myocarditis.

Progressive cardiac fibrosis is a common cause of heart failure. Rho-associated, coiled-coil-containing protein kinases (ROCKs) have been shown to enhance fibrotic processes in the heart and in other organs. In this study, using wild-type, Rock1+/- and Rock2+/- haploinsufficient mice and mouse model of experimental autoimmune myocarditis (EAM) we addressed the role of ROCK1 and ROCK2 in development of myocarditis and postinflammatory fibrosis. We found that myocarditis severity was comparable in wild-type, Rock1+/- and Rock2+/- mice at day 21 of EAM. During the acute stage of the disease, hearts of Rock1+/- mice showed unaffected numbers of CD11b+CD36+ macrophages, CD11b+CD36-Ly6GhiLy6chi neutrophils, CD11b+CD36-Ly6G-Ly6chi inflammatory monocytes, CD11b+CD36-Ly6G-Ly6c- monocytes, CD11b+SiglecF+ eosinophils, CD11b+CD11c+ inflammatory dendritic cells and type I collagen-producing fibroblasts. Isolated Rock1+/- cardiac fibroblasts treated with transforming growth factor-beta (TGF-ß) showed attenuated Smad2 and extracellular signal-regulated kinase (Erk) phosphorylations that were associated with impaired upregulation of smooth muscle actin alpha (αSMA) protein. In contrast to cardiac fibroblasts, expanded Rock1+/- heart inflammatory myeloid cells showed unaffected Smad2 activation but enhanced Erk phosphorylation following TGF-ß treatment. Rock1+/- inflammatory cells responded to TGF-ß by a reduced transcriptional profibrotic response and failed to upregulate αSMA and fibronectin at the protein levels. Unexpectedly, in the EAM model wild-type, Rock1+/- and Rock2+/- mice developed a similar extent of cardiac fibrosis at day 40. In addition, hearts of the wild-type and Rock1+/- mice showed comparable levels of cardiac vimentin, periostin and αSMA. In conclusion, despite the fact that ROCK1 regulates TGF-ß-dependent profibrotic response, neither ROCK1 nor ROCK2 is critically involved in the development of postinflammatory fibrosis in the EAM model.

Identification and Isolation of Cardiac Fibroblasts From the Adult Mouse Heart Using Two-Color Flow Cytometry.

Background: Cardiac fibroblasts represent a main stromal cell type in the healthy myocardium. Activation of cardiac fibroblasts has been implicated in the pathogenesis of many heart diseases. Profibrotic stimuli activate fibroblasts, which proliferate and differentiate into pathogenic myofibroblasts causing a fibrotic phenotype in the heart. Cardiac fibroblasts are characterized by production of type I collagen, but non-transgenic methods allowing their identification and isolation require further improvements. Herein, we present a new and simple flow cytometry-based method to identify and isolate cardiac fibroblasts from the murine heart. Methods and Results: Wild-type and reporter mice expressing enhanced green fluorescent protein (EGFP) under the murine alpha1(I) collagen promoter (Col1a1-EGFP) were used in this study. Hearts were harvested and dissociated into single cell suspensions using enzymatic digestion. Cardiac cells were stained with the erythrocyte marker Ter119, the pan-leukocyte marker CD45, the endothelial cell marker CD31 and gp38 (known also as podoplanin). Fibroblasts were defined in a two-color flow cytometry analysis as a lineage-negative (Lin: Ter119-CD45-CD31-) and gp38-positive (gp38+) population. Analysis of hearts isolated from Col1a1-EGFP reporter mice showed that cardiac Lin-gp38+ cells corresponded to type I collagen-producing cells. Lin-gp38+ cells were partially positive for the mesenchymal markers CD44, CD140a, Sca-1 and CD90.2. Sorted Lin-gp38+ cells were successfully expanded in vitro for up to four passages. Lin-gp38+ cells activated by Transforming Growth Factor Beta 1 (TGF-ß1) upregulated myofibroblast-specific genes and proteins, developed stress fibers positive for alpha smooth muscle actin (αSMA) and showed increased contractility in the collagen gel contraction assay. Conclusions: Two-color flow cytometry analysis using the selected cell surface antigens allows for the identification of collagen-producing fibroblasts in unaffected mouse hearts without using specific reporter constructs. This strategy opens new perspectives to study the physiology and pathophysiology of cardiac fibroblasts in mouse models.

Survival and Functionality of Human Induced Pluripotent Stem Cell-Derived Oligodendrocytes in a Nonhuman Primate Model for Multiple Sclerosis.

: Fast remyelination by endogenous oligodendrocyte precursor cells (OPCs) is essential to prevent axonal and subsequent retrograde neuronal degeneration in demyelinating lesions in multiple sclerosis (MS). In chronic lesions, however, the remyelination capacity of OPCs becomes insufficient. Cell therapy with exogenous remyelinating cells may be a strategy to replace the failing endogenous OPCs. Here, we differentiated human induced pluripotent stem cells (hiPSCs) into OPCs and validated their proper functionality in vitro as well as in vivo in mouse models for MS. Next, we intracerebrally injected hiPSC-derived OPCs in a nonhuman primate (marmoset) model for progressive MS; the grafted OPCs specifically migrated toward the MS-like lesions in the corpus callosum where they myelinated denuded axons. hiPSC-derived OPCs may become the first therapeutic tool to address demyelination and neurodegeneration in the progressive forms of MS. SIGNIFICANCE: This study demonstrates for the first time that human induced pluripotent stem cell (iPSC)-derived oligodendrocyte precursor cells (OPCs), after intracortical implantation in a nonhuman primate model for progressive multiple sclerosis (MS), migrate to the lesions and remyelinate denuded axons. These findings imply that human iPSC-OPCs can be a therapeutic tool for MS. The results of this feasibility study on the potential use of hiPSC-derived OPCs are of great importance for all MS researchers focusing on the stimulation of remyelination in MS patients. Further optimization and research on practical issues related to the safe production and administration of iPSC-derived cell grafts will likely lead to a first clinical trial in a small group of secondary progressive MS patients. This would be the first specific therapeutic approach aimed at restoring myelination and rescuing axons in MS patients, since there is no treatment available for this most debilitating aspect of MS.

Overexpression of polysialylated neural cell adhesion molecule improves the migration capacity of induced pluripotent stem cell-derived oligodendrocyte precursors.

Cell replacement therapy aiming at the compensation of lost oligodendrocytes and restoration of myelination in acquired or congenital demyelination disorders has gained considerable interest since the discovery of induced pluripotent stem cells (iPSCs). Patient-derived iPSCs provide an inexhaustible source for transplantable autologous oligodendrocyte precursors (OPCs). The first transplantation studies in animal models for demyelination with iPSC-derived OPCs demonstrated their survival and remyelinating capacity, but also revealed their limited migration capacity. In the present study, we induced overexpression of the polysialylating enzyme sialyltransferase X (STX) in iPSC-derived OPCs to stimulate the production of polysialic acid-neuronal cell adhesion molecules (PSA-NCAMs), known to promote and facilitate the migration of OPCs. The STX-overexpressing iPSC-derived OPCs showed a normal differentiation and maturation pattern and were able to downregulate PSA-NCAMs when they became myelin-forming oligodendrocytes. After implantation in the demyelinated corpus callosum of cuprizone-fed mice, STX-expressing iPSC-derived OPCs demonstrated a significant increase in migration along the axons. Our findings suggest that the reach and efficacy of iPSC-derived OPC transplantation can be improved by stimulating the OPC migration potential via specific gene modulation.

Generation of induced pluripotent stem cells from hair follicle bulge neural crest stem cells.

Induced pluripotent stem cells (iPSCs) are promising candidates for the study of disease models as well as for tissue engineering purposes. Part of a strategy to develop safe reprogramming technique is reducing the number of exogenous reprogramming factors. Some cells types are more prone to reprogramming than others. iPSC induction with less reprogramming factors has been described in cells with endogenous expression levels of pluripotency genes, such as neural stem cells. Because multipotent neural crest stem cells (NCSCs) from mammalian hair follicle bulges also express pluripotency genes, we argued that this property would facilitate reprogramming of hair follicle bulge NCSCs and could substitute for the use of exogenous reprogramming factors. Although we confirmed the expression of pluripotency genes in hair follicle bulge cells, our results show that these cells do require a full set of reprogramming factors for iPSC induction. Hair follicle bulge-derived iPSCs were created with efficiencies similar to fibroblasts. We conclude that high endogenous levels of pluripotency factors are no guarantee for facilitated induction of pluripotency.