The identity of cells that establish the hematopoietic microenvironment (HME) in human bone marrow (BM), and of clonogenic skeletal progenitors found in BM stroma, has long remained elusive. We show that MCAM/CD146-expressing, subendothelial cells in human BM stroma are capable of transferring, upon transplantation, the HME to heterotopic sites, coincident with the establishment of identical subendothelial cells within a miniature bone organ. Establishment of subendothelial stromal cells in developing heterotopic BM in vivo occurs via specific, dynamic interactions with developing sinusoids. Subendothelial stromal cells residing on the sinusoidal wall are major producers of Angiopoietin-1 (a pivotal molecule of the HSC "niche" involved in vascular remodeling). Our data reveal the functional relationships between establishment of the HME in vivo, establishment of skeletal progenitors in BM sinusoids, and angiogenesis.
Human skeletal progenitors were engineered to stably express R201C mutated, constitutively active Gsα using lentiviral vectors. Long-term transduced skeletal progenitors were characterized by an enhanced production of cAMP, indicating the transfer of the fundamental cellular phenotype caused by activating mutations of Gsα. Like skeletal progenitors isolated from natural FD lesions, transduced cells could generate bone, but not adipocytes or the hematopoietic microenvironment, upon in vivo transplantation. In vitro osteogenic differentiation was noted for the lack of mineral deposition, a blunted up-regulation of osteocalcin, but with enhanced up-regulation of other osteogenic markers, such as ALP and BSP compared to controls. A very potent up-regulation of RANKL expression was observed, which correlates with the pronounced osteoclastogenesis observed in FD lesions in vivo. Stable transduction resulted in a marked up-regulation of selected phosphodiesterase (PDE) isoform mRNAs, and in a prominent increase in total PDE activity. This predicts an adaptive response in skeletal progenitors transduced with constitutively active, mutated Gsα. 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 Gsα, and some of its effects on in vitro differentiation of skeletal progenitors.
Many adenovirus serotypes enter cells by high-affinity binding to the coxsackievirus-adenovirus receptor (CAR) and integrin-mediated internalization. In the present study, we analyzed the possible receptor function of ␣31 for adenovirus serotype 5 (Ad5). We found that penton base and integrin ␣31 could interact in vitro. In vivo, both Ad5-cell binding and virus-mediated transduction were inhibited in the presence of anti-␣3 and anti-1 function-blocking antibodies, and this occurred in both CAR-positive and CAR-negative cell lines. Peptide library screenings and data from binding experiments with wild-type and mutant penton base proteins suggest that the Arg-Gly-Asp (RGD) in the penton base protein, the best known integrin binding motif, is only part of the binding interface with ␣31, which involved multiple additional contact sites.Adenovirus (Ad) host cell entry requires an initial attachment to cells which is mediated by the fiber interaction with the coxsackievirus-Ad receptor (CAR) (2). The subsequent association of the capsid protein penton base with integrin molecules promotes Ad entry (31). Integrins are a family of structurally and functionally related cell surface heterodimeric receptors that mediate cell migration and adhesion. The major extracellular ligands for integrins are collagens, laminins, fibronectin, tenascin, vitronectin, von Willebrand factor, and fibrinogen, reflecting the primary function of integrins in cell adhesion to the extracellular matrix. The ␣v1, -3, -5, -6, and -8 integrins, the ␣51 and ␣81 integrins, and the ␣IIb3 integrins form a subgroup that primarily recognizes ligands containing Arg-Gly-Asp (RGD) motifs (see reference 13 and references therein). Many microorganisms utilize integrins to gain entry into cells: the SA11 rotavirus binds to ␣21 and ␣41 (9), ␣v3 and ␣v1 integrins are receptors of the human parechovirus 1 (30), and ␣v5 has been proposed, although not conclusively, as a coreceptor in adeno-associated virus type 2 infection (27, 29). The foot-and-mouth disease virus uses different integrins for cell infection (14,15,16). Integrin ␣31 is a cellular receptor for Kaposi's sarcoma-associated herpesvirus (1). Yersinia pseudotuberculosis binds to members of the 1 integrin family in order to enter eukaryotic cells (22).Several Ad serotypes contain an RGD motif in the penton base protein. This feature, and the Ad cell-detaching property, suggested an interaction of the virus with the integrin receptors. Indeed, ␣v3 and ␣v5 are receptors for human Ad2 and Ad5, and direct binding to isolated ␣v5 was shown for human Ad2, Ad3, Ad4, Ad5, and Ad37 (24, 31). In hematopoietic and melanoma cells, respectively, the ␣M2 and b1 integrins were found to be implicated in human Ad5 infection (3, 12). More recent evidence indicates ␣v1 as an Ad2 and Ad5 coreceptor in the human embryonic kidney (HEK293) cell line (23). Ad interaction with the ␣v1, -3, and -5 integrin subtypes is efficiently competed by RGD-containing peptides (23, 31). A second integrin binding motif is...
Systems for gene transfer and silencing in human skeletal stem cells (hSSCs, also stromal or mesenchymal stem cells) are important for addressing critical issues in basic hSSC and skeletal biology and for developing gene therapy strategies for treatment of skeletal diseases. Whereas recent studies have shown the efficacy of lentiviral transduction for gene transfer in hSSCs in vitro, no study has yet proven that lentivector-transduced hSSCs retain their distinctive organogenic potential in vivo, as probed by in vivo transplantation assays. Therefore, in addition to analyzing the in vitro growth and differentiation properties of hSSCs transduced with advanced-generation lentivectors, we ectopically transplanted LV-eGFP-transduced hSSCs (along with an osteoconductive carrier) in the subcutaneous tissue of immunocompromised mice. eGFP-transduced cells formed heterotopic ossicles, generating osteoblasts, osteocytes, and stromal cells in vivo, which still expressed GFP at 2 months after transplantation. eGFP-expressing cells could be recovered from the ossicles 8 weeks posttransplantation and reestablished in culture as viable and proliferating cells. Further, we investigated the possibility of silencing individual genes in hSSCs using lentivectors encoding short hairpin precursors of RNA interfering sequences under the control of the Pol-III-dependent H1 promoter. Significant long-term silencing of both lamin A/C and GFP (an endogenous gene and a transgene, respectively) was obtained with lentivectors encoding shRNAs. These data provide the basis for analysis of the effect of gene knockdown during the organogenesis of bone in the in vivo transplantation system and for further studies on the silencing of alleles carrying dominant, disease-causing mutations.
Advances in mechanistic knowledge of human neurological disorders have been hindered by the lack of adequate human in vitro models. Three-dimensional (3D) cellular models displaying higher biological relevance are gaining momentum; however, their lack of robustness and scarcity of analytical tools adapted to three dimensions hampers their widespread implementation. Herein we show that human midbrain-derived neural progenitor cells, cultured as 3D neurospheres in stirred culture systems, reproducibly differentiate into complex tissue-like structures containing functional dopaminergic neurons, as well as astrocytes and oligodendrocytes. Moreover, an extensive toolbox of analytical methodologies has been adapted to 3D neural cell models, allowing molecular and phenotypic profiling and interrogation. The generated neurons underwent synaptogenesis and elicit spontaneous Ca(2+) transients. Synaptic vesicle trafficking and release of dopamine in response to depolarizing stimuli was also observed. Under whole-cell current-and-voltage clamp, recordings showed polarized neurons (Vm=-70 mV) and voltage-dependent potassium currents, which included A-type-like currents. Glutamate-induced currents sensitive to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate antagonists revealed the existence of functional glutamate receptors. Molecular and phenotypic profiling showed recapitulation of midbrain patterning events, and remodeling toward increased similarity to human brain features, such as extracellular matrix composition and metabolic signature. We have developed a robust and reproducible human 3D neural cell model, which may be extended to patient-derived induced pluripotent stem cells, broadening the applicability of this model.
Several studies have demonstrated the potential for vector-mediated gene transfer to the brain. Helper-dependent (HD) human (HAd) and canine (CAV-2) adenovirus, and VSV-G-pseudotyped self-inactivating HIV-1 vectors (LV) effectively transduce human brain cells and their toxicity has been partly analysed. However, their effect on the brain homeostasis is far from fully defined, especially because of the complexity of the central nervous system (CNS). With the goal of dissecting the toxicogenomic signatures of the three vectors for human neurons, we transduced a bona fide human neuronal system with HD-HAd, HD-CAV-2 and LV. We analysed the transcriptional response of more than 47,000 transcripts using gene chips. Chip data showed that HD-CAV-2 and LV vectors activated the innate arm of the immune response, including Toll-like receptors and hyaluronan circuits. LV vector also induced an IFN response. Moreover, HD-CAV-2 and LV vectors affected DNA damage pathways - but in opposite directions - suggesting a differential response of the p53 and ATM pathways to the vector genomes. As a general response to the vectors, human neurons activated pro-survival genes and neuron morphogenesis, presumably with the goal of re-establishing homeostasis. These data are complementary to in vivo studies on brain vector toxicity and allow a better understanding of the impact of viral vectors on human neurons, and mechanistic approaches to improve the therapeutic impact of brain-directed gene transfer.
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