Inflammatory signaling regulates embryonic hematopoietic stem and progenitor cell production.

Identifying signaling pathways that regulate hematopoietic stem and progenitor cell (HSPC) formation in the embryo will guide efforts to produce and expand HSPCs ex vivo. Here we show that sterile tonic inflammatory signaling regulates embryonic HSPC formation. Expression profiling of progenitors with lymphoid potential and hematopoietic stem cells (HSCs) from aorta/gonad/mesonephros (AGM) regions of midgestation mouse embryos revealed a robust innate immune/inflammatory signature. Mouse embryos lacking interferon γ (IFN-γ) or IFN-α signaling and zebrafish morphants lacking IFN-γ and IFN-ϕ activity had significantly fewer AGM HSPCs. Conversely, knockdown of IFN regulatory factor 2 (IRF2), a negative regulator of IFN signaling, increased expression of IFN target genes and HSPC production in zebrafish. Chromatin immunoprecipitation (ChIP) combined with sequencing (ChIP-seq) and expression analyses demonstrated that IRF2-occupied genes identified in human fetal liver CD34(+) HSPCs are actively transcribed in human and mouse HSPCs. Furthermore, we demonstrate that the primitive myeloid population contributes to the local inflammatory response to impact the scale of HSPC production in the AGM region. Thus, sterile inflammatory signaling is an evolutionarily conserved pathway regulating the production of HSPCs during embryonic development.

The composition of the mesenchymal stromal cell compartment in human bone marrow changes during development and aging.

Life-long hematopoiesis depends on the support of mesenchymal stromal cells within the bone marrow. Therefore, changes in the hematopoietic compartment that occur during development and aging probably correlate with variation in the composition of the stromal cell microenvironment. Mesenchymal stromal cells are a heterogeneous cell population and various subtypes may have different functions. In accordance with others, we show that CD271 and CD146 define distinct colony-forming-unit-fibroblast containing mesenchymal stromal cell subpopulations. In addition, analysis of 86 bone marrow samples revealed that the distribution of CD271(bright)CD146(-) and CD271(bright)CD146(+) subsets correlates with donor age. The main subset in adults was CD271(bright)CD146(-), whereas the CD271(bright)CD146(+) population was dominant in pediatric and fetal bone marrow. A third subpopulation of CD271(-)CD146(+) cells contained colony-forming-unit-fibroblasts in fetal samples only. These changes in composition of the mesenchymal stromal cell compartment during development and aging suggest a dynamic system, in which these subpopulations may have different functions.

Cell cycle and tissue of origin contribute to the migratory behaviour of human fetal and adult mesenchymal stromal cells.

Mesenchymal stromal cells (MSC) are potential cells for cellular therapies, in which the recruitment and migration of MSC towards injured tissue is crucial. Our data show that culture-expanded MSC from fetal lung and bone marrow, adult bone marrow and adipose tissue contained a small percentage of migrating cells in vitro, but the optimal stimulus was different. Overall, fetal lung-MSC had the highest migratory capacity. As fetal bone marrow-MSC had lower migratory potential than fetal lung-MSC, the tissue of origin may determine the migratory capacity of MSC. No additive effect in migration towards combined stimuli was observed, which suggests only one migratory MSC fraction. Interestingly, actin rearrangement and increased paxillin phosphorylation were observed in most MSC upon stromal cell-derived factor-1alpha or platelet-derived growth factor-BB stimulation, indicating that this mechanism involved in responding to migratory cues is not restricted to migratory MSC. Migratory MSC maintained differentiation and migration potential, and contained significantly less cells in S- and G2/M-phase than their non-migrating counterpart. In conclusion, our results suggest that MSC from various sources have different migratory capacities, depending on the tissue of origin. Similar to haematopoietic stem cells, cell cycle contributes to MSC migration, which offers perspectives for modulation of MSC to enhance efficacy of future cellular therapies.

Different Balance of Wnt Signaling in Adult and Fetal Bone Marrow-Derived Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) are applied as novel therapeutics for their regenerative and immune-suppressive capacities. Clinical applications, however, require extensive expansion of MSCs. Fetal bone marrow-derived MSCs (FBMSCs) proliferate faster than adult bone marrow-derived MSC (ABMSCs). To optimize expansion and function of MSC in general, we explored the differences between ABMSC and FBMSC. Gene expression profiling implicated differential expression of genes encoding proteins in the Wnt signaling pathway, including excreted inhibitors of Wnt signaling, particularly by ABMSC. Both MSC types had a similar basal level of canonical Wnt signaling. Abrogation of autocrine Wnt production by inhibitor of Wnt production-2 (IWP2) reduced canonical Wnt signaling and cell proliferation of FBMSCs, but hardly affected ABMSC. Addition of exogenous Wnt3a, however, induced expression of the target genes lymphocyte enhancer-binding factor (LEF) and T-cell factor (TCF) faster and at lower Wnt3a levels in ABMSC compared to FBMSC. Medium replacement experiments indicated that ABMSC produce an inhibitor of Wnt signaling that is effective on ABMSC itself but not on FBMSC, whereas FBMSC excrete (Wnt) factors that stimulate proliferation of ABMSC. In contrast, FBMSC were not able to support hematopoiesis, whereas ABMSC displayed hematopoietic support sensitive to IWP2, the inhibitor of Wnt factor excretion. In conclusion, ABMSC and FBMSC differ in their Wnt signature. While FBMSC produced factors, including Wnt signals, that enhanced MSC proliferation, ABMSC produced Wnt factors in a setting that enhanced hematopoietic support. Thus, further unraveling the molecular basis of this phenomenon may lead to improvement of clinical expansion protocols of ABMSCs.

Organ-specific migration of mesenchymal stromal cells: Who, when, where and why?

Mesenchymal stromal cells (MSC) represent a type of multipotent cells that can differentiate to various mesenchymal lineages. MSC can be isolated from different tissues and require ex vivo expansion to exert their regenerative and immunosuppressive function for various clinical applications. The efficacy of these MSC-based therapies at least partly depends on migration and specific homing towards the site where the cells are needed. MSC express a wide variety of integrins, chemokine- and growth factor receptors, though culture-expansion dramatically alters their migratory and engraftment potential. However, it has become clear that tissue damage and/or inflammation can enhance the efficacy of MSC homing. In this review, we focus on the migratory potential of MSC to target organs, including bone marrow, bone, spleen & lymph nodes, intestine and heart, and the underlying molecular mechanisms in various preclinical and clinical settings. Better understanding of directed MSC migration will offer new perspectives to modulate MSC expansion and/or clinical protocols to improve their efficacy upon transplantation.

Articular cartilage endurance and resistance to osteoarthritic changes require transcription factor Erg.

OBJECTIVE: To determine whether and how the transcription factor Erg participates in the genesis, establishment, and maintenance of articular cartilage. METHODS: Floxed Erg mice were mated with Gdf5-Cre mice to generate conditional mutants lacking Erg in their joints. Joints of mutant and control mice were subjected to morphologic and molecular characterization and also to experimental surgically induced osteoarthritis (OA). Gene expression, promoter reporter assays, and gain- and loss-of-function in vitro tests were used to characterize molecular mechanisms of Erg action. RESULTS: Conditional Erg ablation did not elicit obvious changes in limb joint development and overall phenotype in juvenile mice. However, as mice aged, joints of mutant mice degenerated spontaneously and exhibited clear OA-like phenotypic defects. Joints in juvenile mutant mice were more sensitive to surgically induced OA and became defective sooner than operated joints in control mice. Global gene expression data and other studies identified parathyroid hormone-related protein (PTHrP) and lubricin as possible downstream effectors and mediators of Erg action in articular chondrocytes. Reporter assays using control and mutated promoter-enhancer constructs indicated that Erg acted on Ets DNA binding sites to stimulate PTHrP expression. Erg was up-regulated in severely affected areas in human OA articular cartilage but remained barely appreciable in areas of less affected cartilage. CONCLUSION: The study shows for the first time that Erg is a critical molecular regulator of the endurance of articular cartilage during postnatal life and that Erg can mitigate spontaneous and experimental OA. Erg appears to do this through regulating expression of PTHrP and lubricin, factors known for their protective roles in joints.

Mesenchymal stromal cell migration: possibilities to improve cellular therapy.

Mesenchymal stromal cells (MSC) represent a type of multipotent cells that can be isolated from several human tissues and that can be expanded ex vivo for clinical application. The regenerative and immune modulatory capacities of MSC have raised hopes for clinical applications of MSC. At the moment, many clinical trials applying MSC for treatment of multiple diseases are being set up. Currently, extensive expansion (3-6 weeks) is required to obtain enough cells for transplantation. However, culture-expanded MSC have almost completely lost their engraftment potential. MSC expansion cultures are initiated with a heterogeneous, poorly defined cell population. It is unknown which MSC populations are expanded and how this affects homing capacity. Thus, understanding MSC migration will offer perspectives to modulate the expansion protocols to obtain cells that maintain migration and homing capacities. This review highlights our current understanding of MSC migration with particular emphasis on the possibilities to improve MSC-based therapy.

Nuclear receptors Nur77 and Nurr1 modulate mesenchymal stromal cell migration.

Detailed understanding of mesenchymal stromal cells (MSC) migration is imperative for future cellular therapies. To identify genes involved in the process of MSC migration, we generated gene expression profiles of migrating and nonmigrating fetal bone marrow MSC (FBMSC). Only 12 genes showed differential expression in migrating versus nonmigrating FBMSC. The nuclear receptors Nur77 and Nurr1 showed the highest expression in migratory MSC. Nur77 and Nurr1 are members of NR4A nuclear orphan receptor family, and we found that their expression is rapidly increased upon exposure of FBMSC to the migratory stimuli stromal-derived factor-1α (SDF-1α) and platelet-derived growth factor-BB. Lentiviral expression of Nur77 or Nurr1 resulted in enhanced migration of FBMSC toward SDF-1α compared with mock-transduced FBMSC. Analysis of the cell cycle, known to be involved in MSC migration, revealed that expression of Nur77 and Nurr1 decreases the proportion of cells in S-phase compared with control cells. Further, gain-of-function experiments showed increased hepatocyte growth factor expression and interleukin (IL)-6 and IL-8 production in MSC. Despite the altered cytokine profile, FBMSC expressing Nur77 or Nurr1 maintained the capacity to inhibit T-cell proliferation in a mixed lymphocyte reaction. Our results demonstrate that Nur77 and Nurr1 promote FBMSC migration. Modulation of Nur77 and Nurr1 activity may therefore offer perspectives to enhance the migratory potential of FBMSC which may specifically regulate the local immune response.