Human umbilical cord blood-borne fibroblasts contain marrow niche precursors that form a bone/marrow organoid in vivo

Human umbilical cord blood (CB) has attracted much attention as a reservoir for functional hematopoietic stem and progenitor cells, and, recently, as a source of blood-borne fibroblasts (CB-BFs). Previously, we demonstrated that bone marrow stromal cell (BMSC) and CB-BF pellet cultures make cartilage in vitro. Furthermore, upon in vivo transplantation, BMSC pellets remodelled into miniature bone/marrow organoids. Using this in vivo model, we asked whether CB-BF populations that express characteristics of the hematopoietic stem cell (HSC) niche contain precursors that reform the niche. CB ossicles were regularly observed upon transplantation. Compared with BM ossicles, CB ossicles showed a predominance of red marrow over yellow marrow, as demonstrated by histomorphological analyses and the number of hematopoietic cells isolated within ossicles. Marrow cavities from CB and BM ossicles included donor-derived CD146-expressing osteoprogenitors and host-derived mature hematopoietic cells, clonogenic lineage-committed progenitors and HSCs. Furthermore, human CD34+ cells transplanted into ossicle-bearing mice engrafted and maintained human HSCs in the niche. Our data indicate that CB- BFs are able to recapitulate the conditions by which the bone marrow microenvironment is formed and establish complete HSC niches, which are functionally supportive of hematopoietic tissue

Establishment of bone marrow and hematopoietic niches in vivo by reversion of chondrocyte differentiation of human bone marrow stromal cells

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Stem cells and bone diseases: New tools, new perspective

Postnatal skeletal stem cells are a unique class of progenitors with biological properties that extend well beyond the limits of stemness as commonly defined. Skeletal stem cells sustain skeletal tissue homeostasis, organize and maintain the complex architectural structure of the bone marrow microenvironment and provide a niche for hematopoietic progenitor cells. The identification of stem cells in the human post-natal skeleton has profoundly changed our approach to the physiology and pathology of this system. Skeletal diseases have been long interpreted essentially in terms of defective function of differentiated cells and/or abnormal turnover of the matrix that they produce. The notion of a skeletal stem cell has brought forth multiple, novel concepts in skeletal biology that provide potential alternative concepts. At the same time, the recognition of the complex functions played by skeletal progenitors, such as the structural and functional organization of the bone marrow, has provided an innovative, unifying perspective for understanding bone and bone marrow changes simultaneously occurring in many disorders. Finally, the possibility to isolate and highly enrich for skeletal progenitors, enables us to reproduce perfectly normal or pathological organ miniatures. These, in turn, provide suitable models to investigate and manipulate the pathogenetic mechanisms of many genetic and non-genetic skeletal diseases. This article is part of a Special Issue entitled Stem cells and Bone

A unique case of multiple non-ossifying fibromas with polyostotic monomelic distribution and aggressive clinical course.

Multiple non-ossifying fibromas (MNOFs) occur either isolated or in association with other anomalies, are usu- ally localized in the long bones of the lower limbs, may be radiographically confused with other skeletal lesions, and tend to heal spontaneously with the completion of the skeletal growth. Segmental distribution, either monomelic or polymelic and ipsilateral, is rare and commonly observed in the context of developmental diseases known as BRASopathies^, which are caused by mutations in genes that encode components or regulators within the Ras/mitogen- activated protein kinase signaling pathway. We describe here the radiographic and pathologic features of an 18-year-old Caucasian boy, whose clinical history started at the age of 3 when the diagnosis of aneurysmal bone cyst was made on a lytic lesion of his left clavicle. Over the following 2 years, the patient developed polyostotic and monomelic lesions within the left humerus, radius, and ulna. No other skeletal and extra- skeletal anomalies were clinically detected. The lesions were interpreted as consistent with polyostotic fibrous dysplasia and MNOFs and showed an unusually aggressive clinical course with progressive increase in size and coalescence. The definitive diagnosis of MNOFs was made after the exclu- sion of fibrous dysplasia by molecular analysis. The polyostotic and monomelic distribution of the lesions and the unusually aggressive clinical course contribute to make this case of MNOFs unique

Experimental evaluation of sand fly collection and storage methods for the isolation and molecular detection of Phlebotomus-borne viruses

Abstract Background Several viruses have been recently isolated from Mediterranean phlebotomine sand flies; some are known to cause human disease while some are new to science. To monitor the Phlebotomus-borne viruses spreading, field studies are in progress using different sand fly collection and storage methods. Two main sampling techniques consist of CDC light traps, an attraction method allowing collection of live insects in which the virus is presumed to be fairly preserved, and sticky traps, an interception method suitable to collect dead specimens in high numbers, with a risk for virus viability or integrity. Sand flies storage requires a “deep cold chain” or specimen preservation in ethanol. In the present study the influence of sand fly collection and storage methods on viral isolation and RNA detection performances was evaluated experimentally. Methods Specimens of laboratory-reared Phlebotomus perniciosus were artificially fed with blood containing Toscana virus (family Bunyaviridae, genus Phlebovirus). Various collection and storage conditions of blood-fed females were evaluated to mimic field procedures using single and pool samples. Isolation on VERO cell cultures, quantitative Real time-Retro-transcriptase (RT)-PCR and Nested-RT-PCR were performed according to techniques commonly used in surveillance studies. Results Live engorged sand flies stored immediately at −80 °C were the most suitable sample for phlebovirus identification by both virus isolation and RNA detection. The viral isolation rate remained very high (26/28) for single dead engorged females frozen after 1 day, while it was moderate (10/30) for specimens collected by sticky traps maintained up to 3 days at room temperature and then stored frozen without ethanol. Opposed to viral isolation, molecular RNA detection kept very high on dead sand flies collected by sticky traps when left at room temperature up to 6 days post blood meal and then stored frozen in presence (88/95) or absence (87/88) of ethanol. Data were confirmed using sand fly pools. Conclusions While the collection and storage methods investigated had not much impact on the ability to detect viral RNA by molecular methods, they affected the capacity to recover viable viruses. Consequently, sand fly collection and handling procedures should be established in advance depending on the goal of the surveillance studies

Phosphodiestarase-mediatd adaptation to Gsalpha mutations is developmentally regulated in embryonicand post-natal stem cells.

Fibrous dysplasia is a skeletal disorder caused by gain-of-function mutations of Gs-alpha. Although the genetic event occurs in early development, pre-natal skeletal growth is unaffected in FD patients. This suggests that skeletal progenitors display a different sensitivity to aberrant Gs signaling at different developmental ages. To address this issue, we infected mouse embryonic stem cells (ES) and human post-natal skeletal stem cells (Bone Marrow Stromal Cells, BMSCs) with a lentivector expressing the R201C rat Gs-alpha cDNA and analyzed their ability to differentiate into skeletal phenotypes. The expression of the transgene was confirmed at both RNA and protein levels. Surprisingly, neither ES cells nor BMSCs showed excess intracellular cAMP upon infection. However, treatment with IBMX revealed a different response in the two cell types. In transduced BMSCs, IBMX significantly increased intracellular cAMP compared to wild-type cells. Accordingly, high expression of IBMX-sensitive PDE isozymes (i.e. PDE 3, 4 and 7) was detected by q-PCR in basal conditions. IBMX alone did not modify cAMP levels in transgenic ES cells in which increased expression of the IBMX-insensitive PDE 8 was observed. Following incubation in specific inductive media, the expression of cartilage and bone markers was, overall, unaffected in mutated ES cells. Furthermore, mature cartilage undergoing endochondral ossification and normal bone were observed in teratomas generated upon in vivo transplantation. In contrast, differentiation of BMSC was profoundly affected by the mutation. In vitro, abnormal expression of skeletal markers was observed in specific differentiation assays. In vivo, ectopic osteogenic activity was limited to the deposition of a scarce amount of woven bone. In conclusion, these data demonstrate the different behavior of Gs-alpha mutated embryonic vs adult stem cells during skeletal differentiation. Most important, they show that a PDE-mediated adaptive response to Gs-alpha activating mutations operates in both cell types. However, different, and differentially regulated, PDEs isozymes are activated in mutated ES cells compared to postnatal skeletal progenitors, and the resulting adaptive response seems more efficient in ES cells than in postnatal stem cells. This could contribute to explain the normal development and abnormal postnatal growth of FD bone

Transfer, Analysis, and Reversion of the Fibrous Dysplasia Cellular Phenotype in Human Skeletal Progenitors

Human skeletal progenitors were engineered to stably express R201C mutated, constitutively active Gs alpha using lentiviral vectors. Longterm transduced skeletal progenitors were characterized by an enhanced production of cAMP, indicating the transfer of the fundamental cellular phenotype caused by activating mutations of Gs alpha. Like skeletal progenitors isolated from natural fibrous dysplasia (FD) lesions, transduced cells could generate bone but not adipocytes or the hematopoietic microenvironment on in vivo transplantation. In vitro osteogenic differentiation was noted for the lack of mineral deposition, a blunted upregulation of osteocalcin, and enhanced upregulation of other osteogenic markers such as alkaline phosphatase (ALP) and bone sialoprotein (BSP) compared with controls. A very potent upregulation of RANKL expression was observed, which correlates with the pronounced osteoclastogenesis observed in FD lesions in vivo. Stable transduction resulted in a marked upregulation of selected phosphodiesterase (PDE) isoform mRNAs and a prominent increase in total PDE activity. This predicts an adaptive response in skeletal progenitors transduced with constitutively active, mutated Gs alpha. Indeed, like measurable cAMP levels, the differentiative responses of transduced skeletal progenitors were profoundly affected by inhibition of PDEs or lack thereof. Finally, using lentiviral vectors encoding short hairpin (sh) RNA interfering sequences, we demonstrated that selective silencing of the mutated allele is both feasible and effective in reverting the aberrant cAMP production brought about by the constitutively active Gs alpha and some of its effects on in vitro differentiation of skeletal progenitors. (C) 2010 American Society for Bone and Mineral Research

HIV Tat-mediated transcriptional regulation of proteasome protein cleavage specificity

The major antigen-adapted immune response protecting a vertebrate against virus infection is that mediated by CTLs (cytotoxic T-lymphocytes). CTLs destroy virus-infected cells, thereby containing the infection. They are activated by recognition of peptide antigens or epitopes, presented to them in the context of MHC I proteins. These epitopes are derived from proteolytic degradation of endogenously synthesized proteins, which is mediated by the proteasome. Augmentation of epitope presentation by MHC I is thought to be effected by the immunoproteasome, induced in response to IFN-γ (interferon-γ) in some cells, and constitutively expressed in others. In this issue of the Biochemical Journal, Remoli and colleagues describe the manipulation of the immunoproteasome by the Tat (transcriptional activation) protein of HIV. The authors show that Tat deregulates the balance of the three proteins, LMP2 (low-molecular-mass polypeptide 2), LMP7 and MECL1 (multicatalytic endopeptidase complex-like 1), which distinguish the immunoproteasome from the proteasome, and they provide a molecular explanation. Intracellular Tat sequesters IRF-1 (interferon-regulatory factor-1) from its cognate promoter element, where normally it associates with STAT1 (signal transducer and activator of transcription 1) to activate basal transcription of the LMP2 gene. LMP2 expression is inhibited as a consequence, skewing the stoichiometry of the immunoproteasome and changing its enzymatic activity. These findings provide a molecular account of an immunomodulatory activity of HIV: changing the peptide antigen profile of cells expressing or exposed to Tat. They may also provide an avenue for manipulating vaccine efficacy and specificity with Tat-based adjuvants

Moderated poster - AVNRT cryoablation in children < 26 kg: efficacy and safety of electrophysiologically guided low voltage bridge strategy

Background and Aim: Recent studies have reported that the low voltage bridge (LVB) strategy is very effective in atrioventricular nodal reentrant tachycardia (AVNRT) ablation. The purpose of our study was to evaluate the efficacy and safety of AVNRT cryoablation in pediatric patients &lt; 26 Kg using the electrophysiologically guided LVB strategy both in the acute phase and in a mid-term follow up. Methods: Thirteen pediatric patients (69% males, mean age 7±1.65 years, mean weight 24.8±1.77 Kg), with previous history of AVNRT confirmed by the transesophageal atrial stimulation, underwent slow pathway cryoablation using the electrophysiologically guided LVB strategy. Results: In 12 patients a typical AVNRT was induced, while 1 patient showed both a typical and an atypical form. An AV “jump” occurred in 46% of patients. One small LVB was found in 7 patients (54%) at the M1-M2 level, in 4 (31%) at P2 or P2-M1 level, in 1(7.5%) at A2 and another 1 (7.5%) at M1 level. Acute success was 100% with an average of 5 cryoablations per patient. No peri-procedural complications occurred. All procedures were performed with near-zero fluoroscopy. No recurrence occurred during the follow up (10.58±10.23 months). Conclusion: AVNRT cryoablation using the electrophysiologically guided LVB strategy seems to be an effective and very safe procedure in small children who are the highest risk patients for this type of ablation