Sally Boxall

Markers for Characterization of Bone Marrow Multipotential Stromal Cells

Given the observed efficacy of culture-expanded multipotential stromal cells, also termed mesenchymal stem cells (MSCs), in the treatment of graft-versus host and cardiac disease, it remains surprising that purity and potency characterization of manufactured cell batches remains rather basic. In this paper, we will initially discuss surface and molecular markers that were proposed to serve as the indicators of the MSC potency, in terms of their proliferative potential or the ability to differentiate into desired lineages. The second part of this paper will be dedicated to a critical discussion of surface markers of uncultured (i.e., native) bone marrow (BM) MSCs. Although no formal consensus has yet been reached on which markers may be best suited for prospective BM MSC isolation, markers that cross-react with MSCs of animal models (such as CD271 and W8-B2/MSCA-1) may have the strongest translational value. Whereas small animal models are needed to discover the in vivo function on these markers, large animal models are required for safety and efficacy testing of isolated MSCs, particularly in the field of bone and cartilage tissue engineering.

Transcriptional profile of native CD271+ multipotential stromal cells: Evidence for multiple fates, with prominent osteogenic and Wnt pathway signaling activity

OBJECTIVE Controversy surrounds the identity and functionality of rare bone marrow-derived multipotential stromal cells (BM-MSCs), including their differentiation capabilities, their relationship to pericytes and hematopoiesis-supporting stromal cells, and the relevance of their culture-expanded progeny in studies of skeletal biology and development of cell-based therapies. The aim of this study was to clarify the nature of candidate BM-MSCs by profiling transcripts that reflect different aspects of their putative functions in vivo. METHODS Rare, sorted BM-derived CD45(-/low) CD271(bright) (CD271) cells were analyzed using 96-gene expression arrays focused on transcripts relevant to mesenchymal-lineage differentiation (toward bone, cartilage, fat, or muscle), hematopoietic and stromal support, and molecules critical to skeletal homeostasis. These cells were compared to matched CD45+ CD271- hematopoietic-lineage cells, culture-expanded MSCs, and skin fibroblasts. When feasible, transcription was validated using flow cytometry. RESULTS CD271 cells had a transcriptional profile consistent with the multiple fates of in vivo MSCs, evident from the observed simultaneous expression of osteogenic, adipogenic, pericytic, and hematopoiesis-supporting genes (e.g., SP7 [osterix], FABP4 [fatty acid binding protein 4], ANGPT1 [angiopoietin 1], and CXCL12 [stromal cell-derived factor 1], respectively). Compared to culture-expanded MSCs and fibroblasts, CD271 cells exhibited greater transcriptional activity, particularly with respect to Wnt-related genes (>1,000-fold increased expression of FRZB [secreted frizzled-related protein 3] and WIF1 [Wnt inhibitory factor 1]). A number of transcripts were identified as novel markers of MSCs. CONCLUSION The native, BM-derived in vivo MSC population is endowed with a gene signature that is compatible with multiple functions, reflecting the topographic bone niche of these cells, and their signature is significantly different from that of culture-expanded MSCs. This indicates that studies of the biologic functions of MSCs in musculoskeletal diseases, including osteoporosis and osteoarthritis, should focus on in vivo MSCs, rather than their culture-adapted progeny.

High abundance of CD271 + multipotential stromal cells (MSCs) in intramedullary cavities of long bones

Aspiration of iliac crest bone marrow (ICBM) remains the most frequent technique used in harvesting multipotential stromal cells (MSCs) for bone regeneration. Although this tissue type is easily accessed by a surgeon, it has a low frequency of MSCs, which is significant given the high cell numbers required for bone regeneration strategies. Lipoaspirates possess higher MSC frequencies, albeit cells with a differentiation profile less suited to orthopaedic interventions. Intra-medullary cavities of long bones have previously been shown to harbour MSCs in animals, however evaluation of their frequency, differentiation capacity and phenotype in humans had not previously been performed. Long bone fatty bone marrow (LBFBM) was collected prior to harvesting bone graft. Basic cellular compositions of donor-matched LBFBM and ICBM aspirates, including the numbers of CD34+ hematopoietic stem cells and CD31+ endothelial cells, were similar. MSCs were enumerated using colony-forming-unit-fibroblast assays and flow cytometry for the presence of a resident LBFBM CD45−/low CD271+ MSC population and revealed a trend for higher MSC numbers (average 5 fold, n = 6) per millilitre of LBFBM compared to donor-matched ICBM. Functional characteristics of resident MSCs, including their growth rates, differentiation potentials and surface phenotypes (CD73+CD105+CD90+) before and after culture-amplification, were similar. Enhanced numbers of MSCs could be recovered following brief enzymatic treatment of solid fragments of LBFBM. Our findings therefore reveal that the intramedullary cavity of the human femur is a depot of MSCs, which, although closely associated with fat, have a differentiation profile equivalent to ICBM. This anatomical site is frequently accessed by the orthopaedic/trauma surgeon and aspiration of the intramedullary cavity represents a ‘low-tech’ method of harvesting potentially large numbers of MSCs for regenerative therapies and research. This article is part of a Special Issue entitled: Interactions Between Bone, Adipose Tissue and Metabolism.

Gastric Helicobacter infection induces iron deficiency in the INS-GAS mouse.

There is increasing evidence from clinical and population studies for a role of H. pylori infection in the aetiology of iron deficiency. Rodent models of Helicobacter infection are helpful for investigating any causal links and mechanisms of iron deficiency in the host. The aim of this study was to investigate the effects of gastric Helicobacter infection on iron deficiency and host iron metabolism/transport gene expression in hypergastrinemic INS-GAS mice. INS-GAS mice were infected with Helicobacter felis for 3, 6 and 9 months. At post mortem, blood was taken for assessment of iron status and gastric mucosa for pathology, immunohistology and analysis of gene expression. Chronic Helicobacter infection of INS- GAS mice resulted in decreased serum iron, transferrin saturation and hypoferritinemia and increased Total iron binding capacity (TIBC). Decreased serum iron concentrations were associated with a concomitant reduction in the number of parietal cells, strengthening the association between hypochlorhydria and gastric Helicobacter-induced iron deficiency. Infection with H. felis for nine months was associated with decreased gastric expression of iron metabolism regulators hepcidin, Bmp4 and Bmp6 but increased expression of Ferroportin 1, the iron efflux protein, iron absorption genes such as Divalent metal transporter 1, Transferrin receptor 1 and also Lcn2 a siderophore-binding protein. The INS-GAS mouse is therefore a useful model for studying Helicobacter-induced iron deficiency. Furthermore, the marked changes in expression of gastric iron transporters following Helicobacter infection may be relevant to the more rapid development of carcinogenesis in the Helicobacter infected INS-GAS model.

Haematopoietic repopulating activity in human cord blood CD133 + quiescent cells

We have demonstrated previously that cord blood CD133+ cells isolated in the G0 phase of the cell cycle are highly enriched for haematopoietic stem cell (HSC) activity, in contrast to CD133+G1 cells. Here, we have analysed the phenotype and functional properties of this population in more detail. Our data demonstrate that a large proportion of the CD133+G0 cells are CD38 negative (60.4%) and have high aldehyde dehydrogenase activity (75.1%) when compared with their CD133+G1 counterparts (13.5 and 4.1%, respectively). This suggests that stem cell activity resides in the CD133+G0 population. In long-term BM cultures, the CD133+G0 cells generate significantly more progenitors than the CD34+G0 population (P<0.001) throughout the culture period. Furthermore, a comparison of CD133+G0 versus CD133+G1 cells revealed that multilineage reconstitution was obtained only in non-obese diabetic/SCID animals receiving G0 cells. We conclude that CD133+ cells in the quiescent phase of the cell cycle have a phenotype consistent with HSCs and are highly enriched for repopulating activity when compared with their G1 counterparts. This cell population should prove useful for selection and manipulation in ex vivo expansion protocols.

Intrinsic multipotential mesenchymal stromal cell activity in gelatinous Heberden’s nodes in osteoarthritis at clinical presentation

IntroductionGelatinous Heberden’s nodes (HNs), also termed synovial cysts, are a common form of generalized osteoarthritis (OA). We sought to determine whether HN cases at clinical presentation contained multipotential stromal cells (MSCs) and to explore whether such cells were more closely related to bone marrow (BM) or synovial fluid (SF) MSCs by transcriptional analysis.MethodsAt clinical presentation, gelatinous material was extracted/extruded from the distal phalangeal joint of OA patients with HNs. From this, plastic adherent cells were culture-expanded for phenotypic and functional characterization and comparison with BM- and SF-MSCs. Mesenchymal related gene expression was studied by using a custom-designed TaqMan Low Density Array to determine transcriptional similarities between different MSC groups and skin fibroblasts.ResultsIn all cases, HN material produced MSC-like colonies. Adherent cultures displayed an MSC phenotype (CD29+, CD44+, CD73+, CD81+, and CD90+ and CD14- CD19-, CD31-, CD34-, CD45-, and HLADR-) and exhibited osteogenic, chondrogenic lineage differentiation but weak adipogenesis. Gene cluster analysis showed that HN-MSCs were more closely related to SF- than normal or OA BM-MSCs with significantly higher expression of synovium-related gene markers such as bone morphogenic protein 4 (BMP4), bone morphogenetic protein receptor type 1A (BMPR1A), protein/leucine-rich end leucine-rich repeat protein (PRELP), secreted frizzled-related protein 4 (SFRP4), and tumor necrosis factor alpha-induced protein 6 (TNFAIP6) (P <0.05).ConclusionsGelatinous HNs derived from hand OA at clinical presentation contain a population of MSCs that share transcriptional similarities with SF-derived MSCs. Their aberrant entrapment within the synovial cysts may impact on their normal role in joint homeostasis.

The use of multiparameter flow cytometry and cell sorting to characterize native human bone marrow mesenchymal stem cells (MSC).

This chapter describes a method for identification, phenotypic analysis, and cell sorting of rare mesenchymal stem cells (MSCs) from human bone marrow (BM) aspirates. The native BM MSC population is identified based on the CD45(-/low)CD271(+) phenotype. The method consists of three related procedures: Procedure 1 involves a microbead-based pre-enrichment step. Two other procedures describe direct flow cytometric analysis of MSCs following the isolation of the mononuclear cell (MNC) fraction (Procedure 2) or more rapidly, following a simple ammonium chloride-based red cell lysis (Procedure 3). Recently described multi-lineage transcript expression in the CD45(-/low)CD271(+) cells suggests that the native BM MSC fraction could be further subdivided into functionally distinct subpopulations. The present protocols are hoped to help MSC biologists to enter this exciting field of research and to take it forward towards a better understanding of MSC biology in vivo.

T1633 Gastric Helicobacter Infection Induces Iron Deficiency in the INS-GAS Mouse

There is increasing evidence from clinical and population studies for a role of H. pylori infection in the aetiology of iron deficiency. Rodent models of Helicobacter infection are helpful for investigating any causal links and mechanisms of iron deficiency in the host. The aim of this study was to investigate the effects of gastric Helicobacter infection on iron deficiency and host iron metabolism/transport gene expression in hypergastrinemic INS-GAS mice. INS-GAS mice were infected with Helicobacter felis for 3, 6 and 9 months. At post mortem, blood was taken for assessment of iron status and gastric mucosa for pathology, immunohistology and analysis of gene expression. Chronic Helicobacter infection of INS- GAS mice resulted in decreased serum iron, transferrin saturation and hypoferritinemia and increased Total iron binding capacity (TIBC). Decreased serum iron concentrations were associated with a concomitant reduction in the number of parietal cells, strengthening the association between hypochlorhydria and gastric Helicobacter-induced iron deficiency. Infection with H. felis for nine months was associated with decreased gastric expression of iron metabolism regulators hepcidin, Bmp4 and Bmp6 but increased expression of Ferroportin 1, the iron efflux protein, iron absorption genes such as Divalent metal transporter 1, Transferrin receptor 1 and also Lcn2 a siderophore-binding protein. The INS-GAS mouse is therefore a useful model for studying Helicobacter-induced iron deficiency. Furthermore, the marked changes in expression of gastric iron transporters following Helicobacter infection may be relevant to the more rapid development of carcinogenesis in the Helicobacter infected INS-GAS model.

A4.08 Predicting senescence-related loss of MSC osteogenic capacity irrespective of donor age

Background and objectives Multipotential stromal cells (MSCs) have considerable regenerative capabilities and their premature senescence in vivo may be responsible for altered bone phenotypes in age-related skeletal diseases including osteoporosis and osteoarthritis. Following minimal culture expansion, MSCs lose their native molecular phenotype (Churchman, Arthritis Rheum, 2012). However it is not known whether this continues to change as MSC reach senescence and whether any molecular markers could be predictive of senescence-related loss of MSC osteogenic capacity. Materials and methods Bone marrow (BM, n = 7) was obtained from a deliberately diverse age range (2–72 years) of healthy donors. MSCs were selected for by plastic adherence and culture expanded up to senescence. Throughout the expansion period samples were taken for RNA and gDNA extraction as well as osteogenic differentiation. Gene expression profiles were studied by 96-gene Taqman low density array, whilst epigenetic senescence signature analysis was performed on the gDNA. Results Cultures from older donors reached senescence considerably earlier compared to younger donors (r = -0.703). For all donors combined, MSC osteogenic capacity correlated better with population doublings (PDs) before senescence than with accrued PDs. Regardless of the donor age, the majority (59/78) of the genes were unchanged throughout the expansion period, with exceptions including TNFRSF11B/osteoprotegrin, CDH11/osteogenic cadherin, COL1A1/collagen type I and SPARC/osteonectin (all higher by senescence compared to early passage), and NANOG (lower in senescent cultures). The methylation status of cells correlated equally well with both PD and PD before senescence. Following osteogenic differentiation many genes were upregulated up to 200-fold and TNFRSF11B was less upregulated closer to senescence. Conclusion Compared to methylation markers, the expression of TNFRSF11B/osteoprotegrin may be a better predictor of BM MSC senescence and related loss of osteogenic capability independent of donor age.

THU0030 Evidence for good intrinsic mesenchymal stem cell activity in gelatinous heberden’s nodes in osteoarthritis at clinical presentation

Background Heberden’s nodes (HN) are common pre-radiographic features of generalised osteoarthritis (GOA) and they may present as acute cystic lesions before eventually leading to florid new bone formation [1]. Objectives To examine if the gelatinous material from Heberden’s nodes “synovial cysts” contained multipotential mesenchymal stromal cells (MSCs), and whether such cells were of bone marrow or synovial fluid origin and to link the findings to joint structure especially articular cartilage integrity. Methods Two patients with clinical OA having Heberden’s nodes in their distal interphalangeal (DIP) joint at clinical presentation were imaged with conventional x-ray and high resolution MRI to ascertain the extent of joint involvement. Gelatinous material from the synovial cysts was extruded and plastic-adherent cells were expanded in MSC conditions and characterised phenotypically and functionally utilising trilineage differentiation assays. Mesenchymal related gene expression was studied using a Custom Taqman Low Density Array (69 Genes) to determine the underlying origin and molecular profile of the cells. Results Imaging showed that one case had excellent articular cartilage preservation but had osteophyte formation, whilst the other case had more extensive joint damage. The HN material formed MSC-like colonies, displayed an MSC phenotype being negative for CD14, CD19, CD31, CD34, CD45 and HLADR, and positive for CD29, CD44, CD73, CD90, CD105 and CD166. Functionally these cells exhibited osteogenic, adipogenic and chondrogenic lineage differentiation. Gene cluster analysis showed that HN-MSCs were more closely related to synovial fluid (SF-) than to bone marrow (BM-MSCs) with much higher expression of SFRP4 (Secreted Frizzled-Related Protein 4), a newly identified synovium/SF-MSC marker [2]. Conclusions This is the first description of a resident population of MSCs in a small joint in which, we show their presence in acute Heberden’s nodes. The presence of these MSCs even in cases where there is good preservation of articular cartilage indicates Heberden’s node stem cell activity may be a key early event in hand OA pathogenesis. References Kellgren JH, Moore R. Generalized osteoarthritis and Heberden’s nodes. Br Med J 1952;1:181-7. Sekiya I, Ojima Ml. Human mesenchymal stem cells in synovial fluid increase in the knee with degenerated cartilage and osteoarthritis. J Orthop Res 2011. Epub 2011/12/08. Disclosure of Interest None Declared