Heterotopic bone formation derived from multipotent stromal cells is not inhibited in aged mice.

BACKGROUND AIMS: Decreased bone formation with age is believed to arise, at least in part, because of the influence of the senescent microenvironment. In this context, it is unclear whether multipotent stromal cell (MSC)-based therapies would be effective for the treatment of bone diseases. METHODS: With the use of a heterotopic bone formation model, we investigated whether MSC-derived osteogenesis is impaired in aged mice compared with young mice. RESULTS: We found that bone formation derived from MSCs is not reduced in aged mice. These results are supported by the unexpected finding that conditioned media collected from ionizing radiation-induced senescent MSCs can stimulate mineralization and delay osteoclastogenesis in vitro. CONCLUSIONS: Overall, our results suggest that impaired bone formation with age is mainly cell-autonomous and provide a rationale for the use of MSC-based therapies for the treatment of bone diseases in the elderly.

Loss of the osteogenic differentiation potential during senescence is limited to bone progenitor cells and is dependent on p53.

DNA damage can lead to the induction of cellular senescence. In particular, we showed that exposure to ionizing radiation (IR) leads to the senescence of bone marrow-derived multipotent stromal cells (MSC) and osteoblast-like stromal cells (OB-SC), a phenotype associated with bone loss. The mechanism by which IR leads to bone dysfunction is not fully understood. One possibility involves that DNA damage-induced senescence limits the regeneration of bone progenitor cells. Another possibility entails that bone dysfunction arises from the inability of accumulating senescent cells to fulfill their physiological function. Indeed, we show here that exposure to IR prevented the differentiation and mineralization functions of MSC, an effect we found was limited to this population as more differentiated OB-SC could still form mineralize nodules. This is in contrast to adipogenesis, which was inhibited in both IR-induced senescent MSC and 3T3-L1 pre-adipocytes. Furthermore, we demonstrate that IR-induced loss of osteogenic potential in MSC was p53-dependent, a phenotype that correlates with the inability to upregulate key osteogenic transcription factors. These results are the first to demonstrate that senescence impacts osteogenesis in a cell type dependent manner and suggest that the accumulation of senescent osteoblasts is unlikely to significantly contribute to bone dysfunction in a cell autonomous manner.

Mapping and kinetics of microglia/neuron cell-to-cell contacts in the 6-OHDA murine model of Parkinson's disease.

As neuroinflammatory processes are involved in the pathogenesis of Parkinson's disease (PD), we provide several key data describing the time-course of microglial accumulation in relation with behavioral alterations and neurodegeneration in a murine model of PD induced by intrastriatal injection of 6-hydroxydopamine (6-OHDA). Our study argues for a major role of microglia which accumulation is somehow early and transient in spite of the neuronal loss progression. Moreover, we observed less 6-OHDA-induced neurodegeneration associated with less inflammatory reaction in DAP-12 Knock-In mice. The direct cell-to-cell contacts that may support physical interactions between microglia and altered dopaminergic neurons are ill-defined, while it is currently hypothesized that microglia support an immune-mediated amplification of neurodegeneration by establishing a molecular cross talk with neurons. Indeed, we sought to map microglia/neuron appositions in substantia nigra (SN) of 6-OHDA injected C57Bl/6 mice and CX3CR1/(GFP/+) mice. Confocal immunofluorescence analyses followed by 3D reconstitutions reveal close appositions between the soma of TH+ neurons and microglial cell bodies and ramifications. Interestingly, some microglial ramifications penetrated TH(+) somas and about 40% of GFP(+) microglial cells in the injured SN harbored TH(+) intracytoplasmic inclusions. These results suggest a direct cross talk between neurons and microglia that may exert a microphagocytic activity toward TH+ neurons. Altogether, these results obtained in a murine PD model may participate in the understanding of microglial cells' function in neurodegenerative diseases.

DAP12 overexpression induces osteopenia and impaired early hematopoiesis.

ITAM-bearing transmembrane signaling adaptors such as DAP12 and FcRγ are important players in bone homeostasis, but their precise role and functions are still unknown. It has been shown that osteoclast differentiation results from the integration of the RANK and of the DAP12 and FcRγ signaling pathways. DAP12-deficient mice suffer from a mild osteopetrosis and culture of their bone marrow cells in the presence of M-CSF and RANKL, fails to give rise to multinucleated osteoclasts. Here, we report that mice overexpressing human DAP12 have an osteopenic bone phenotype due to an increased number of osteoclasts on the surface of trabecular and cortical bone. This enhanced number of osteoclasts is associated with an increased number of proliferating myeloid progenitors in Tg-hDAP12 mice. It is concomitant with an arrest of B cell development at the Pre-Pro B/Pre B stage in the bone marrow of Tg-hDAP12 mice and important decrease of follicular and marginal B cells in the spleen of these animals. Our data show that the overexpression of DAP12 results in both increased osteoclastogenesis and impaired hematopoiesis underlining the relationship between bone homeostasis and hematopoiesis.

Therapeutic efficacy of cord blood-derived mesenchymal stromal cells for the prevention of acute graft-versus-host disease in a xenogenic mouse model.

Human mesenchymal stromal cells (MSCs) have been successfully utilized for the treatment of refractory graft-versus-host disease (GvHD). Despite the large number of in vitro and in vivo models developed for clarifying their immunomodulatory properties, the mechanism of action of MSCs remains elusive and their efficacy controversial. Here, we tested the ability of cord blood-derived MSCs to alleviate the symptoms of GvHD induced by the injection of human peripheral blood mononuclear cells into NOD/SCID/γc(-) mice. In this in vivo xeno-GvHD model, we demonstrate that a single MSC injection is able to inhibit GvHD in terms of clinical signs and related mortality. We also show that in this model MSCs act by both immunomodulating T-cells and fostering recovery after irradiation. The translational impact of these findings could provide a reliable preclinical model for studying the efficacy, dosage, and time of administration of human MSCs for the prevention of acute GvHD.

Ionizing radiation-induced expression of INK4a/ARF in murine bone marrow-derived stromal cell populations interferes with bone marrow homeostasis.

Alterations of the BM microenvironment have been shown to occur after chemoradiotherapy, during aging, and after genetic manipulations of telomere length. Nevertheless, whether BM stromal cells adopt senescent features in response to these events is unknown. In the present study, we provide evidence that exposure to ionizing radiation (IR) leads murine stromal BM cells to express senescence markers, namely senescence-associated ß-galactosidase and increased p16(INK4a)/p19(ARF) expression. Long (8 weeks) after exposure of mice to IR, we observed a reduction in the number of stromal cells derived from BM aspirates, an effect that we found to be absent in irradiated Ink4a/arf-knockout mice and to be mostly independent of the CFU potential of the stroma. Such a reduction in the number of BM stromal cells was specific, because stromal cells isolated from collagenase-treated bones were not reduced after IR. Surprisingly, we found that exposure to IR leads to a cellular nonautonomous and Ink4a/arf-dependent effect on lymphopoiesis. Overall, our results reveal the distinct sensitivity of BM stromal cell populations to IR and suggest that long-term residual damage to the BM microenvironment can influence hematopoiesis in an Ink4a/arf-dependent manner.

Bidirectional interactions between bone metabolism and hematopoiesis.

Interactions between hematopoiesis and bone metabolism have been described in various developmental and pathological situations. Here we review this evidence from the literature with a focus on microenvironmental regulation of hematopoiesis and bone metabolism. Our hypothesis is that this process occurs by bidirectional signaling between hematopoietic and mesenchymal cells through cell adhesion molecules, membrane-bound growth factors, and secreted matrix proteins. Examples of steady-state hematopoiesis and pathologies are presented and support our view that hematopoietic and mesenchymal cell functions are modulated by specific microenvironments in the bone marrow.

Gene signature of stromal cells which support dendritic cell development.

Spleen stromal cells are critical determinants of dendritic cell (DC) development in spleen. The spleen stromal line, namely STX3, supports DC differentiation in vitro from overlaid bone marrow cells while the lymph node stromal line, namely 2RL22, does not. Here we have characterised the hematopoietic support capacity of each stroma, and analysed lineage origin of the stromal cell lines by gene profiling using microarrays. Stromal co-culture experiments were performed using bone marrow cells as a source of hematopoietic progenitors. A characteristic immature myeloid-like CD11c(+)CD11b(+)CD86(+)MHC-II(/lo)B220()CD8alpha() DC is produced after 14 days in STX3 cocultures, while 2RL22 cocultures produce only monocyte/macrophage-like cells. No other hematopoietic cell type is produced. The STX3 and 2RL22 stroma were compared by transcriptome analysis utilising Affymetrix Murine U74Av2 genechips to identify gene expression related to differential hematopoietic support function. Data mining was used to determine cell surface marker expression reflecting endothelial cells and fibroblasts, as well as adhesion molecules contributing to the microenvironment. STX3 shows gene expression reflective of early endothelial cells, while 2RL22 expresses markers specific to fibroblasts. The expression of genes like Flt1, CD34, Mcam, and Eng distinguishes STX3 as an early immature endothelial cell lacking markers of angioblasts or hemangioblasts like Tal1/SCL, Tie1, Tie2, Kdr or Prom1/AC133. The absence of expression of genes like Vwf and Cd31 distinguishes STX3 from fully differentiated vascular endothelial cells. In contrast, the 2RL22 lymph node stroma specifically expresses genes related to fibroblastic-like cells like osteoblasts with expression of Vdr (Vitamin D receptor), and epithelial cells with expression of Krt13 (keratins). Gene expression data identifies STX3 as splenic endothelial cells, independently able to support the outgrowth of immature, myeloid DC-like cells from progenitors present in bone marrow, while 2RL22 lymph node fibroblastic cells provide support for development of monocytes/macrophages.

Use of gene profiling to describe a niche for dendritic cell development.

Gene profiling provides a multitude of data on individual gene expression. The view is expressed here that unreplicated data can be used in a descriptive way to compare cell populations in terms of their lineage characteristics and function. In these studies, the aim is to provide a snapshot of gene expression or its absence as a reflection of cell lineage or type, rather than gain a reliable expression measure for all genes expressed. The data set used in this analysis represents gene expression in the splenic stroma STX3 supportive of dendritic cell hematopoiesis and the lymph node stroma 2RL22, which is non-supportive. These were obtained by hybridization of Affymetrix U74Av2 genechips. The use of P-value selection to identify genes with a high probability of differential expression has been used effectively to detect differentially expressed genes. Genes that relate to a niche environment for hematopoiesis have been selected for further study to make predictions about the cell types of supportive stroma.

The role of stroma in hematopoiesis and dendritic cell development.

Development of the immune system is depicted as a hierarchical process of differentiation from hematopoietic stem cells (HSC) to lineage-committed precursors, which further develop into mature immune cells. In the case of dendritic cell (DC) development, this linear precursor-progeny approach has led to a confused picture of relationships between various subsets of DC identifiable in vivo. A possible reconciliation of the diversity of DC precursors and DC subsets in vivo encompasses the role of the microenvironment in DC hematopoiesis. We propose here that various niches for DC hematopoiesis within lymphoid organs could account for the diversity of DC in vivo. A tridimensional space consisting of stromal cells which produce a range of membrane-bound and secreted molecules providing signals to DC progenitors would define these niches.

Heterogeneity amongst splenic stromal cell lines which support dendritic cell hematopoiesis.

Long-term cultures (LTC) producing dendritic cells (DC) have been previously established from spleen. LTC support the development of nonadherent cells comprising small DC progenitors and immature DC. Similarly, the splenic stroma STX3, derived from a LTC which ceased DC production, can support DC development from precursors in overlaid bone marrow. The STX3 stroma is an immortalised mixed population of endothelial cells and elongated spindle-shaped cells, thought to be fibroblasts. The stromal cell components of STX3 have been studied here. A panel of 102 cell lines was established by single-cell sorting. A range of clone morphology, including cobblestone cells and elongated spindle-shaped cells, was reflective of heterogeneity in STX3. However, similar expression levels for the endothelial genes ACVRL1/ ALK1, COL18A1, and MCAM in 13 splenic stromal cell lines suggested that both cell types had endothelial origin. The hematopoietic support function of stromal clones was tested in coculture assays with a bone marrow cell overlay. Splenic stromal cell lines with different morphology were both supporters and nonsupporters of hematopoiesis, in terms of foci formation or release of suspension cells. Cloning of STX3 led to the isolation of a panel of splenic endothelial cell lines heterogeneous in terms of morphology and hematopoietic support function.

Splenic endothelial cell lines support development of dendritic cells from bone marrow.

Although growth factors are commonly used to generate dendritic cells (DCs) in vitro, the role of the microenvironment necessary for DC development is still poorly understood. The mixed splenic stromal cell population STX3 defines an in vitro microenvironment supportive of DC development. Dissection of cellular components of the STX3 stroma should provide information about a niche for DC development. STX3 was therefore cloned by single-cell sorting, and a panel of 102 splenic stromal cell lines was established. Four representative splenic stromal cell lines that support hematopoiesis from bone marrow are described here in terms of stromal cell type and DC production. All four stromal lines express the endothelial genes Acvrl1, Cd34, Col18a1, Eng, Flt1, Mcam, and Vcam1 but not Cd31 or Vwf. Three of the four lines form tube-like structures when cultured on Matrigel. Their endothelial maturity correlates with the ability to support myeloid DC development from bone marrow. A fourth cell line, unable to form tube-like structures in Matrigel, produced large granulocytic cells expressing CD11b and CD86 but not CD11c and CD80. Conditioned media from splenic stromal cell lines also support DC production, indicating that soluble growth factors and cytokines produced by stromal lines drive DC development. This article reports characterization of immature endothelial cell lines derived from spleen that are supportive of DC development and predicts the existence of such a cell type in vivo which regulates DC development within spleen.

Molecular definition of an in vitro niche for dendritic cell development.

OBJECTIVE: Although dendritic cell (DC) precursors have been isolated from many lymphoid sites, the regulation and location of early DC development is still poorly understood. Here we describe a splenic microenvironment that supports DC hematopoiesis in vitro and identify gene expression specific for that niche. METHODS: The DC supportive function of the STX3 splenic stroma and the lymph node-derived 2RL22 stroma for overlaid bone marrow cells was assessed by coculture over 2 weeks. The DC supportive function of SXT3 was identified in terms of specific gene expression in STX3 and not 2RL22 using Affymetrix microchips. RESULTS: STX3 supports DC differentiation from overlaid bone marrow precursors while 2RL22 does not. A dataset of 154 genes specifically expressed in STX3 and not 2RL22 was retrieved from Affymetrix results. Functional annotation has led to selection of 26 genes as candidate regulators of the microenvironment supporting DC hematopoiesis. Specific expression of 14 of these genes in STX3 and not 2RL22 was confirmed by reverse transcription-polymerase chain reaction. CONCLUSION: Some genes specifically expressed in STX3 have been previously associated with hematopoietic stem cell niches. A high proportion of genes encode growth factors distinct from those commonly used for in vitro development of DC from precursors. Potential regulators of a DC microenvironment include genes involved in angiogenesis, hematopoiesis, and development, not previously linked to DC hematopoiesis.

Dendritic cell development in long-term spleen stromal cultures.

The cellular microenvironments in which dendritic cells (DCs) develop are not known. DCs are commonly expanded from CD34+ bone marrow precursors or blood monocytes using a cocktail of growth factors including GM-CSF. However, cytokine-supported cultures are not suitable for studying the intermediate stages of DC development, since progenitors are quickly driven to become mature DCs that undergo limited proliferation and survive for only a short period of time. This lab has developed a long-term culture (LTC) system from spleen which readily generates a high yield of DCs. Hematopoietic cells develop under more normal physiological conditions than in cultures supplemented with cytokines. A spleen stromal cell monolayer supports stem cell maintenance, renewal, and the specific differentiation of only DCs and no other hematopoietic cells. Cultures maintain continuous production of a small population of small-sized progenitors and a large population of fully developed DCs. Cell-cell interaction between stromal cells and progenitor cells is critical for DC differentiation. The progenitors maintained in LTC appear to be quite distinct from bone marrow-derived DC progenitors that respond to GM-CSF. The majority of cells produced in LTC are large-sized cells with a phenotype reflecting myeloid-like DC precursors or immature DCs. These cells are highly endocytotic and weakly immunostimulatory for T cells. This model system predicts in situ production of DCs in spleen from endogenous progenitors, as well as a central role for spleen in DC hematopoiesis.

A role for niches in the development of a multiplicity of dendritic cell subsets.

Although most studies on murine dendritic cell (DC) differentiation concentrate on the nature of the DC precursor population and the lineage relationship between DC and other hematopoietic cell types, very little research addresses the nature of the microenvironments necessary for DC hematopoiesis. Evidence supporting a major contribution of niches in DC differentiation within hematopoietic tissues is reviewed. A model is presented that identifies a potential role for multiple hematopoietic niches in DC differentiation. It is proposed that multiple DC subsets develop from one or a small number of DC progenitor types that lodge in various niches within different tissue sites. Implications of a niche-mediated model for differentiation of DC precursors are discussed in the context of both physiological and pathological situations.

Matrix protein mutations contribute to inefficient induction of apoptosis leading to persistent infection of human neural cells by vesicular stomatitis virus.

In a model system to study factors involved in the establishment of a persistent viral infection that may lead to neurodegenerative diseases, Indiana and New Jersey variants of vesicular stomatitis virus (VSV) with different capacities to infect and persist in human neural cells were studied. Indiana matrix (M) protein mutants and the wild-type New Jersey strain persisted in the human neural cell line H4 for at least 120 days. The Indiana wild-type virus (HR) and a non-M mutant (TP6), both unable to persist, induced apoptosis more strongly than all the other variants tested, as indicated by higher levels of DNA fragmentation and caspase-3-like activity. Transfection of H4 cells with mRNA coding for the VSV M protein confirmed the importance of this protein in the induction of apoptosis. Furthermore, the pan-caspase inhibitor ZVAD-fmk maintained cell survival to about 80%, whereas inhibition of caspase-8, caspase-9, or both only partially protected the cells against death, consistent with the fact that anti-apoptotic molecules from the Bcl-2 family also protect cells from death only partially. These results suggest that VSV activates many pathways of cell death and that an inefficient induction of caspase-3-related apoptosis participates in the establishment of a persistent infection of human neural cells by less virulent VSV variants.