Large-scale extraction and characterization of CD271+ multipotential stromal cells from trabecular bone in health and osteoarthritis: implications for bone regeneration strategies based on uncultured or minimally cultured multipotential stromal cells.

OBJECTIVE: To test the hypothesis that CD45(low)CD271+ bone marrow multipotential stromal cells (MSCs) are abundant in the trabecular bone niche and to explore their functional "fitness" in health and osteoarthritis (OA). METHODS: Following enzymatic extraction, MSC release was evaluated using colony-forming unit-fibroblast (CFU-F) and colony-forming unit-osteoblast assays, flow cytometry, and confocal microscopy. CD45(low)CD271+ cells isolated by fluorescence-activated cell sorting were enumerated and expanded under standard and clonal conditions. Their proliferative and osteogenic potencies were assessed in relation to donor age and compared with those of aspirated CD45(low)CD271+ cells. In vitro and in vivo MSC "aging" was measured using quantitative polymerase chain reaction-based telomere length analysis, and standard differentiation assays were utilized to demonstrate multipotentiality. RESULTS: Cellular isolates from trabecular bone cavities contained approximately 65-fold more CD45(low)CD271+ cells compared with aspirates (P < 0.0001) (median 1.89% [n = 39] and 0.029% [n = 46], respectively), concordant with increased CFU-F release. Aspirated and enzymatically released CD45(low)CD271+ cells had identical MSC phenotypes (approximately 100% CD73+CD105+CD13+, approximately 50-60% CD146+CD106+CD166+) and contained large proportions of highly clonogenic multipotential cells. In vitro osteogenic potency of freshly isolated CD45(low)CD271+ cells was comparable with, and often above, that of early-passage MSCs (8-14%). Their frequency and in vivo telomere status in OA bone were similar to those in bone from age-matched controls. CONCLUSION: Our findings show that CD45(low)CD271+ MSCs are abundant in the trabecular bone cavity and indistinguishable from aspirated CD45(low)CD271+ MSCs. In OA they display aging-related loss of proliferation but no gross osteogenic abnormality. These findings offer new opportunities for direct study of MSCs in musculoskeletal diseases without the requirement for culture expansion. They are also relevant for direct therapeutic exploitation of prospectively isolated, minimally cultured MSCs in trauma and OA.

Mesenchymal stem cells in rheumatoid synovium: enumeration and functional assessment in relation to synovial inflammation level.

OBJECTIVE: Achieving joint regeneration in rheumatoid arthritis (RA) represents a future challenge. Autologous synovial mesenchymal stem cells (MSCs) could be therapeutically exploited. However, the inflammatory milieu in the RA synovium could adversely affect endogenous MSC function. To test this hypothesis, the frequency and multipotency of RA synovial MSCs was evaluated in relation to existing synovial inflammation. METHODS: Synovial inflammation was measured using the arthroscopic visual analogue score (VAS) and further validated using immunohistochemistry and flow cytometry. Highly proliferative clonogenic in vivo MSCs were enumerated following fluorescence-activated cell sorting and expansion for 20 population doublings. MSC multipotency was quantified following standard in vitro culture expansion and trilineage differentiation assays. Real-time PCR, flow cytometry and ELISA were used to evaluate pro- and anti-chondrogenic molecules in standard polyclonal synovial MSCs. RESULTS: The arthroscopic VAS significantly correlated with synovial macrophage infiltration. In RA, synovial MSC chondrogenesis was inhibited in direct relation to VAS (r = -0.777, p<0.05) and reduced compared with control osteoarthritis (OA)-MSCs (p<0.05). In vivo, MSCs resided in the synovial fibroblastic/stromal fraction (CD45(-)CD31(-)) and were reduced in frequency in relation to VAS (r = -0.695, p<0.05). In RA-MSCs, CD44 levels correlated negatively with inflammation and positively with chondrogenesis (r = -0.830 and r = 0.865, respectively). Cytokine production and Sox9 expression was similar in RA-MSCs and OA-MSCs. CONCLUSIONS: There is a negative relationship between synovial MSC chondrogenic and clonogenic capacities and the magnitude of synovitis in RA. Effective suppression of joint inflammation is therefore necessary for the development of autologous MSC treatments aimed at cartilage regeneration in RA.

Regulation of Angiogenesis Discriminates Tissue Resident MSCs from Effective and Defective Osteogenic Environments.

BACKGROUND: The biological mechanisms that contribute to atrophic long bone non-union are poorly understood. Multipotential mesenchymal stromal cells (MSCs) are key contributors to bone formation and are recognised as important mediators of blood vessel formation. This study examines the role of MSCs in tissue formation at the site of atrophic non-union. MATERIALS AND METHODS: Tissue and MSCs from non-union sites (n = 20) and induced periosteal (IP) membrane formed following the Masquelet bone reconstruction technique (n = 15) or bone marrow (n = 8) were compared. MSC content, differentiation, and influence on angiogenesis were measured in vitro. Cell content and vasculature measurements were performed by flow cytometry and histology, and gene expression was measured by quantitative polymerase chain reaction (qPCR). RESULTS: MSCs from non-union sites had comparable differentiation potential to bone marrow MSCs. Compared with induced periosteum, non-union tissue contained similar proportion of colony-forming cells, but a greater proportion of pericytes (p = 0.036), and endothelial cells (p = 0.016) and blood vessels were more numerous (p = 0.001) with smaller luminal diameter (p = 0.046). MSCs showed marked differences in angiogenic transcripts depending on the source, and those from induced periosteum, but not non-union tissue, inhibited early stages of in vitro angiogenesis. CONCLUSIONS: In vitro, non-union site derived MSCs have no impairment of differentiation capacity, but they differ from IP-derived MSCs in mediating angiogenesis. Local MSCs may thus be strongly implicated in the formation of the immature vascular network at the non-union site. Attention should be given to their angiogenic support profile when selecting MSCs for regenerative therapy.

A novel TNFRSF1A splice mutation associated with increased nuclear factor kappaB (NF-kappaB) transcription factor activation in patients with tumour necrosis factor receptor associated periodic syndrome (TRAPS).

OBJECTIVE: To characterise and investigate the functional consequences of a novel TNFRSF1A splice site mutation causing tumour necrosis factor receptor associated periodic syndrome (TRAPS) in a 16-year-old male patient and his mother. METHODS: Mutational DNA screening was performed in the patient and his mother. Western blotting was used to analyse protein expression levels of TNFR1. A multiplex bead immunoassay was used to quantify serum levels of range of cytokines, and an ELISA-based transcription factor assay to measure nuclear factor (NF)-kappaB transactivation. Serum levels of soluble TNFR1 (sTNFR1) were measured by ELISA and fluorescence-activated cell sorting (FACS) analysis used to measure monocyte TNFR1 cell surface expression. RESULTS: A novel mutation, c.472+1G>A (C158delinsYERSSPEAKPSPHPRG), involving a splice site in intron 4 of TNFRSF1A, was found in the proband and affected mother leading to a 45 nucleotide insertion of intronic DNA into the mRNA, resulting in an in-frame insertion of 15 amino acids in the mature TNFR1 protein and a deletion of a cysteine residue C129 (158) in cysteine rich domain (CRD)3. The patients had reduced serum sTNFR1 and surface expression levels of TNFR1, with marked increases in pro- and anti-inflammatory cytokine. Their peripheral blood mononuclear cells (PBMC) had increased basal NF-kappaB activation compared with healthy controls and also had increased p50 nuclear expression following tumour necrosis factor (TNF) stimulation compared with PBMC from healthy controls, as well as T50M (T79M) and C88R (C117R) patients with TRAPS and patients with rheumatoid arthritis (RA). CONCLUSION: A novel, TRAPS causing, TNFRSF1A splice site mutation is associated with decreased sTNFR1 levels, cell surface and whole cell extract expression and increased NF-kappaB transcription factor activation.

Interleukin-7 in rheumatoid arthritis.

Recent data from several groups demonstrate high levels of IL-7 in the joints of RA patients, but much lower levels in OA. In contrast, circulating levels of IL-7 in RA remain a point of debate. IL-7 has many roles in T cell, dendritic cell and bone biology in humans. Reduced levels of circulating IL-7 probably underlie a number of the dysfunctions associated with circulating T cells in RA and may provide a mechanism for some of the unexplained systemic manifestations of the disease. However, IL-7 in the joint may have a more sinister role, contributing to a vicious cycle perpetuating inflammation. Typically, IL-1beta and TNF-alpha increase the stromal production of IL-7 and in turn, IL-7 up-regulates the production of TNF-alpha by macrophages. Most importantly, IL-7 induces the production of osteoclastogenic cytokines by T cells, leading to the maturation of osteoclasts and therefore bone destruction. By linking the stroma with innate and adaptive immunity in RA, IL-7 may be directing the cellular network, leading to chronic inflammation and joint destruction. Blocking IL-7 may well therefore be of therapeutic value.