Activated fibronectin-secretory phenotype of mesenchymal stromal cells in pre-fibrotic myeloproliferative neoplasms.

We characterized bone marrow stromal cells (BMSC) from patients with pre-fibrotic myeloproliferative neoplasms (MPN). MPN-BMSC showed decreased capacity to stimulate the proliferation of colony-forming units of normal hematopoietic stem and progenitor cells and displayed increased matrix remodelling (in particular fibronectin deposition) compared to control BMSC. This finding was confirmed in pre-fibrotic MPN bone marrow biopsies in a tissue microarray (n = 34), which stained positive for fibronectin in the absence of reticulin as a standard myelofibrosis marker. Fibronectin expression correlated significantly with reduced haemoglobin levels in MPN-patients (p = 0.007; R2 = 0.42). Our data show significant cell-intrinsic alterations in MPN-MSC and suggest that Fibronectin expression might be applicable as a biomarker for the identification of early myelofibrotic transformation in reticulin-negative MPN.

Cell fusion enhances mesendodermal differentiation of human induced pluripotent stem cells.

Human induced pluripotent stem cells (iPS cells) resemble embryonic stem cells and can differentiate into cell derivatives of all three germ layers. However, frequently the differentiation efficiency of iPS cells into some lineages is rather poor. Here, we found that fusion of iPS cells with human hematopoietic stem cells (HSCs) enhances iPS cell differentiation. Such iPS hybrids showed a prominent differentiation bias toward hematopoietic lineages but also toward other mesendodermal lineages. Additionally, during differentiation of iPS hybrids, expression of early mesendodermal markers-Brachyury (T), MIX1 Homeobox-Like Protein 1 (MIXL1), and Goosecoid (GSC)-appeared with faster kinetics than in parental iPS cells. Following iPS hybrid differentiation there was a prominent induction of NODAL and inhibition of NODAL signaling blunted mesendodermal differentiation. This indicates that NODAL signaling is critically involved in mesendodermal bias of iPS hybrid differentiation. In summary, we demonstrate that iPS cell fusion with HSCs prominently enhances iPS cell differentiation.

Osteogenesis of heterotopically transplanted mesenchymal stromal cells in rat models of chronic kidney disease.

The current study is based on the hypothesis of mesenchymal stromal cells (MSCs) contributing to soft-tissue calcification and ectopic osteogenesis in chronic kidney disease (CKD). Rat MSCs were transplanted intraperitoneally in an established three-dimensional collagen-based model in healthy control animals and two rat models of CKD and vascular calcification: (1) 5/6 nephrectomy + high phosphorus diet; and (2) adenine nephropathy. As internal controls, collagen gels without MSCs were transplanted in the same animals. After 4 and 8 weeks, MSCs were still detectable and proliferating in the collagen gels (fluorescence-activated cell sorting [FACS] analysis and confocal microscopy after fluorescence labeling of the cells). Aortas and MSC-containing collagen gels in CKD animals showed distinct similarities in calcification (micro-computed tomography [µCT], energy-dispersive X-ray [EDX] analysis, calcium content), induction of osteogenic markers, (ie, bone morphogenic protein 2 [BMP-2], Runt related transcription factor 2 [Runx2], alkaline phosphatase [ALP]), upregulation of the osteocytic marker sclerostin and extracellular matrix remodeling with increased expression of osteopontin, collagen I/III/IV, fibronectin, and laminin. Calcification, osteogenesis, and matrix remodeling were never observed in healthy control animals and non-MSC-containing collagen gels in all groups. Paul Karl Horan 26 (PKH-26)-labeled, 3G5-positive MSCs expressed Runx2 and sclerostin in CKD animals whereas PKH-26-negative migrated cells did not express osteogenic markers. In conclusion, heterotopically implanted MSCs undergo osteogenic differentiation in rat models of CKD-induced vascular calcification, supporting our hypothesis of MSCs as possible players in heterotopic calcification processes of CKD patients.

Novel insights into osteogenesis and matrix remodelling associated with calcific uraemic arteriolopathy.

BACKGROUND: Calcific uraemic arteriolopathy (CUA) or calciphylaxis is a rare, life-threatening disease predominantly occurring in patients with end-stage renal disease. Its pathogenesis has been suggested to include ectopic osteogenesis in soft tissue and the vasculature associated with extracellular matrix (ECM) remodelling. METHODS: To gain further insights into the pathogenesis of CUA, we performed systematic analyses of skin specimens obtained from seven CUA patients including histology, immunohistochemistry, electron microscopy, electron dispersive X-ray analysis (EDX) and quantitative real-time RT-PCR. Skin specimens of (i) seven patients without chronic kidney disease and without CUA and (ii) seven dialysis patients without CUA served as controls. RESULTS: In the CUA skin lesions, we observed a significant upregulation of bone morphogenic protein 2 (BMP-2), its target gene Runx2 and its indirect antagonist sclerostin. Furthermore, we detected an increased expression of inactive uncarboxylated matrix Gla protein (Glu-MGP). The upregulation of osteogenesis-associated markers was accompanied by an increased expression of osteopontin, fibronectin, laminin and collagen I indicating an extensive remodelling of the subcutaneous ECM. EDX analysis revealed calcium/phosphate accumulations in the subcutis of all CUA patients with a molar ratio of 1.68 ± 0.06 matching that of hydroxyapatite mineral. Widespread media calcification in cutaneous arterioles was associated with destruction of the endothelial layer and partial exfoliation of the endothelial cells (ECs). CD31 immunostaining revealed aggregates of ECs contributing to intraluminal obstruction and consecutive malperfusion resulting in the clinical picture of ulcerative necrosis in all seven patients. CONCLUSIONS: Our data indicate that CUA is an active osteogenic process including the upregulation of BMP-2 signalling, hydroxyapatite deposition and extensive matrix remodelling of the subcutis.

Use of multilocus methylation-specific single nucleotide primer extension (MS-SNuPE) technology in diagnostic testing for human imprinted loci.

A number of diseases have been found to be linked to aberrant methylation of specific genes. However, most of the routine diagnostic techniques to detect epigenetic disturbances are restricted to single loci. Additionally, a precise quantification of the methylation status is often hampered. A considerable fraction of patients with Silver-Russell syndrome, Beckwith-Wiedemann syndrome and transient neonatal diabetes mellitus exhibit loss of methylation at further imprinted loci in addition to the disease specific ones (multilocus methylation defects, MLMD). As the currently available tests are mainly focused on single imprinted loci on different chromosomes and thereby make the detection of multilocus methylation defects time-consuming and expensive, we established methylation-specific single nucleotide primer extension (MS-SNuPE) assays for a simultaneous quantification of methylation at multiple methylated loci. We chose loci generally affected in patients with MLMD. The method was validated by screening 66 individuals with known (epi)genetic disturbances. In comparison to other methylation-specific techniques, multilocus methylation-specific single nucleotide primer extension allows the quantitative analysis of numerous CpG islands of different loci in one assay and is, therefore, suitable for the simultaneous diagnostic testing for different congenital imprinting disorders in parallel, as well as for MLMD.

3D co-culture of hematopoietic stem and progenitor cells and mesenchymal stem cells in collagen scaffolds as a model of the hematopoietic niche.

Here, we propose a collagen-based three-dimensional (3D) environment for hematopoietic stem and progenitor cells (HPC) with mesenchymal stem cells (MSC) derived either from bone marrow (BM) or umbilical cord (UC), to recapitulate the main components of the BM niche. Mechanisms described for HPC homeostasis were systematically analyzed in comparison to the conventional liquid HPC culture. The 3D-cultivation allows dissecting two sub-populations of HPC: (I) HPC in suspension above the collagen gel and (II) migratory HPC in the collagen fibres of the collagen gel. The different sites represent distinct microenvironments with significant impact on HPC fate. HPC in niche I (suspension) are proliferative and a dynamic culture containing HPC (CD34(+)/CD38(-)), maturing myeloid cells (CD38(+), CD13(+), CAE(+)) and natural killer (NK) cells (CD56(+)). In contrast, HPC in niche II showed clonal growth with significant high levels of the primitive CD34(+)/CD38(-) phenotype with starting myeloid (CD13(+), CAE(+)) differentiation, resembling the endosteal part of the BM niche. In contrast, UC-MSC are not adequate for HSC expansion as they significantly enhance HPC proliferation and lineage commitment. In conclusion, the 3D-culture system using collagen and BM-MSC enables HPC expansion and provides a potential platform to dissect regulatory mechanisms in hematopoiesis.

Disturbed methylation at multiple imprinted loci: an increasing observation in imprinting disorders.

The widely accepted association between aberrant methylation at specific imprinted loci and distinct imprinting disorders has recently been brought into question by the identification of methylation defects at multiple loci (multilocus methylation defect [MLMD]). Strikingly, in different imprinting disorders, the same MLMD patterns can be observed. The cause for this ambiguous epigenotype-phenotype correlation is currently unknown. Future strategies to solve this enigma have to include all levels of imprinting regulation, ranging from DNA methylation to chromatin organization, as any disturbance of the balanced interaction between the different players in imprinting regulation might cause disturbed expression of imprinted factors. The molecular analysis of MLMD will help in discovering these interactions and contribute to the understanding of genomic imprinting and its disturbances.