IL-17A and TNF Modulate Normal Human Spinal Entheseal Bone and Soft Tissue Mesenchymal Stem Cell Osteogenesis, Adipogenesis, and Stromal Function.

OBJECTIVE: The spondylarthritides (SpA) are intimately linked to new bone formation and IL-17A and TNF pathways. We investigated spinal soft tissue and bone mesenchymal stem cell (MSC) responses to IL-17A and TNF, including their osteogenesis, adipogenesis, and stromal supportive function and ability to support lymphocyte recruitment. METHODS: Normal spinal peri-entheseal bone (PEB) and entheseal soft tissue (EST) were characterized for MSCs by immunophenotypic, osteogenic, chondrogenic, and adipogenic differentiation criteria. Functional and gene transcriptomic analysis was carried out on undifferentiated, adipo- differentiated, and osteo-differentiated MSCs. The enthesis C-C Motif Chemokine Ligand 20-C-C Motif Chemokine Receptor 6 (CCL20-CCR6) axis was investigated at transcript and protein levels to ascertain whether entheseal MSCs influence local immune cell populations. RESULTS: Cultured MSCs from both PEB and EST displayed a tri-lineage differentiation ability. EST MSCs exhibited 4.9-fold greater adipogenesis (p < 0.001) and a 3-fold lower osteogenic capacity (p < 0.05). IL-17A induced greater osteogenesis in PEB MSCs compared to EST MSCs. IL-17A suppressed adipogenic differentiation, with a significant decrease in fatty acid-binding protein 4 (FABP4), peroxisome proliferator-activated receptor gamma (PPARγ), Cell Death Inducing DFFA Like Effector C (CIDEC), and Perilipin-1 (PLIN1). IL-17A significantly increased the CCL20 transcript (p < 0.01) and protein expression (p < 0.001) in MSCs supporting a role in type 17 lymphocyte recruitment. CONCLUSIONS: Normal spinal enthesis harbors resident MSCs with different in vitro functionalities in bone and soft tissue, especially in response to IL-17A, which enhanced osteogenesis and CCL20 production and reduced adipogenesis compared to unstimulated MSCs. This MSC-stromal-enthesis immune system may be a hitherto unappreciated mechanism of "fine tuning" tissue repair responses at the enthesis in health and could be relevant for SpA understanding.

Genomic prediction of agronomic traits in wheat using different models and cross-validation designs.

KEY MESSAGE: Genomic predictions across environments and within populations resulted in moderate to high accuracies but across-population genomic prediction should not be considered in wheat for small population size. Genomic selection (GS) is a marker-based selection suggested to improve the genetic gain of quantitative traits in plant breeding programs. We evaluated the effects of training population (TP) composition, cross-validation design, and genetic relationship between the training and breeding populations on the accuracy of GS in spring wheat (Triticum aestivum L.). Two populations of 231 and 304 spring hexaploid wheat lines that were phenotyped for six agronomic traits and genotyped with the wheat 90 K array were used to assess the accuracy of seven GS models (RR-BLUP, G-BLUP, BayesB, BL, RKHS, GS + de novo GWAS, and reaction norm) using different cross-validation designs. BayesB outperformed the other models for within-population genomic predictions in the presence of few quantitative trait loci (QTL) with large effects. However, including fixed-effect marker covariates gave better performance for an across-population prediction when the same QTL underlie traits in both populations. The accuracy of prediction was highly variable based on the cross-validation design, which suggests the importance to use a design that resembles the variation within a breeding program. Moderate to high accuracies were obtained when predictions were made within populations. In contrast, across-population genomic prediction accuracies were very low, suggesting that the evaluated models are not suitable for prediction across independent populations. On the other hand, across-environment prediction and forward prediction designs using the reaction norm model resulted in moderate to high accuracies, suggesting that GS can be applied in wheat to predict the performance of newly developed lines and lines in incomplete field trials.

Evidence for the Accumulation of Nonsynonymous Mutations and Favorable Pleiotropic Alleles During Wheat Breeding.

Plant breeding leads to the genetic improvement of target traits by selecting a small number of genotypes from among typically large numbers of candidate genotypes after careful evaluation. In this study, we first investigated how mutations at conserved nucleotide sites normally viewed as deleterious, such as nonsynonymous sites, accumulated in a wheat, Triticum aestivum, breeding lineage. By comparing a 150 year old ancestral and modern cultivar, we found recent nucleotide polymorphisms altered amino acids and occurred within conserved genes at frequencies expected in the absence of purifying selection. Mutations that are deleterious in other contexts likely had very small or no effects on target traits within the breeding lineage. Second, we investigated if breeders selected alleles with favorable effects on some traits and unfavorable effects on others and used different alleles to compensate for the latter. An analysis of a segregating population derived from the ancestral and modern parents provided one example of this phenomenon. The recent cultivar contains the Rht-B1b green revolution semi-dwarfing allele and compensatory alleles that reduce its negative effects. However, improvements in traits other than plant height were due to pleiotropic loci with favorable effects on traits and to favorable loci with no detectable pleiotropic effects. Wheat breeding appears to tolerate mutations at conserved nucleotide sites and to only select for alleles with both favorable and unfavorable effects on traits in exceptional situations.

The Transcriptional Landscape of Polyploid Wheats and Their Diploid Ancestors during Embryogenesis and Grain Development.

Modern wheat production comes from two polyploid species, Triticum aestivum and Triticum turgidum (var durum), which putatively arose from diploid ancestors Triticum urartu, Aegilops speltoides, and Aegilops tauschii How gene expression during embryogenesis and grain development in wheats has been shaped by the differing contributions of diploid genomes through hybridization, polyploidization, and breeding selection is not well understood. This study describes the global landscape of gene activities during wheat embryogenesis and grain development. Using comprehensive transcriptomic analyses of two wheat cultivars and three diploid grasses, we investigated gene expression at seven stages of embryo development, two endosperm stages, and one pericarp stage. We identified transcriptional signatures and developmental similarities and differences among the five species, revealing the evolutionary divergence of gene expression programs and the contributions of A, B, and D subgenomes to grain development in polyploid wheats. The characterization of embryonic transcriptional programming in hexaploid wheat, tetraploid wheat, and diploid grass species provides insight into the landscape of gene expression in modern wheat and its ancestral species. This study presents a framework for understanding the evolution of domesticated wheat and the selective pressures placed on grain production, with important implications for future performance and yield improvements.plantcell;31/12/2888/FX1F1fx1.

Comparative study of biological characteristics of mesenchymal stem cells isolated from mouse bone marrow and peripheral blood.

Mesenchymal stromal cells (MSCs) possess self-renewal and multilineage differentiation potential, indicating their prospects as cellular therapeutic agents for regenerative medicine. Although adult bone marrow (BM) is the major source of these cells for clinical use, harvesting requires invasive procedures. Therefore, alternative sources, such as peripheral blood (PB), are needed. The objective of the current study was to compare PB-MSCs and BM-MSCs with regard to their biological characteristics. PB-MSCs and BM-MSCs were isolated from 4-week-old BALB/c white mice by density gradient centrifugation and cultured in DMEM + 10% fetal bovine serum until passage four. Morphological features, proliferation, cell surface marker expression and trilineage differentiation potential were assessed for both PB-MSCs and BM-MSCs. No significant differences in morphological features were observed. BM-MSCs had a higher proliferative capability than PB-MSCs as measured by XTT assays. Both PB-MSCs and BM-MSCs had broadly similar cell surface marker expression, but PB-MSCs had positive expression of cluster of differentiation (CD)146 and CD140b. Both PB-MSCs and BM-MSCs were capable of trilineage differentiation. Although BM-MSCs had a greater capacity for osteogenic and chondrogenic differentiation than PB-MSCs, PB-MSCs had a better capability for adipogenic differentiation than BM-MSCs. In conclusion, PB-MSCs and BM-MSCs have very similar biological characteristics. Thus, PB is a promising source for easily obtaining MSCs in mice.

The novel cytokine Metrnl/IL-41 is elevated in Psoriatic Arthritis synovium and inducible from both entheseal and synovial fibroblasts.

Meteorin-like(IL-41), is a novel cytokine that is thought to be immunoregulatory and is highly expressed in psoriatic skin. We investigated IL-41 protein expression in synovial tissue in RA(Rheumatoid Arthritis), PsA(Psoriatic Arthritis) and OA(Osteoarthritis) patients and evaluated IL-41 production from healthy enthesis samples, as the enthesis represent the primary inflammatory site in PsA. IL-41 was measured in synovial fluid from PsA, RA and OA patients. Synovial biopsies were stained for IL-41 by immunohistochemistry. IL-41 was highly expressed in the synovial fluid and synovial tissue of PsA patients (median = 7722 pg/ml) when compared to OA patients (median = 5044 pg/ml). We found that entheseal stromal cells were the dominant producer of IL-41 from the enthesis. Moreover, stromal derived IL-41, could be further induced by IL-17A/F and TNF. In conclusion, IL-41 is expressed in PsA synovium and is present and inducible at the enthesis. Its functional effect in psoriatic inflammation remains to be fully elucidated.

Identification of senescent cells in multipotent mesenchymal stromal cell cultures: Current methods and future directions.

Regardless of their tissue of origin, multipotent mesenchymal stromal cells (MSCs) are commonly expanded in vitro for several population doublings to achieve a sufficient number of cells for therapy. Prolonged MSC expansion has been shown to result in phenotypical, morphological and gene expression changes in MSCs, which ultimately lead to the state of senescence. The presence of senescent cells in therapeutic MSC batches is undesirable because it reduces their viability, differentiation potential and trophic capabilities. Additionally, senescent cells acquire senescence-activated secretory phenotype, which may not only induce apoptosis in the neighboring host cells following MSC transplantation, but also trigger local inflammatory reactions. This review outlines the current and promising new methodologies for the identification of senescent cells in MSC cultures, with a particular emphasis on non-destructive and label-free methodologies. Technologies allowing identification of individual senescent cells, based on new surface markers, offer potential advantage for targeted senescent cell removal using new-generation senolytic agents, and subsequent production of therapeutic MSC batches fully devoid of senescent cells. Methods or a combination of methods that are non-destructive and label-free, for example, involving cell size and spectroscopic measurements, could be the best way forward because they do not modify the cells of interest, thus maximizing the final output of therapeutic-grade MSC cultures. The further incorporation of machine learning methods has also recently shown promise in facilitating, automating and enhancing the analysis of these measured data.

Exome sequencing highlights the role of wild-relative introgression in shaping the adaptive landscape of the wheat genome.

Introgression is a potential source of beneficial genetic diversity. The contribution of introgression to adaptive evolution and improvement of wheat as it was disseminated worldwide remains unknown. We used targeted re-sequencing of 890 diverse accessions of hexaploid and tetraploid wheat to identify wild-relative introgression. Introgression, and selection for improvement and environmental adaptation, each reduced deleterious allele burden. Introgression increased diversity genome wide and in regions harboring major agronomic genes, and contributed alleles explaining a substantial proportion of phenotypic variation. These results suggest that historic gene flow from wild relatives made a substantial contribution to the adaptive diversity of modern bread wheat.

Identification of myeloid cells in the human enthesis as the main source of local IL-23 production.

OBJECTIVE: We investigated whether the normal human spinal enthesis contained resident myeloid cell populations, capable of producing pivotal proinflammatory cytokines including tumour necrosis factor (TNF) and interleukin (IL)-23 and determined whether these could be modified by PDE4 inhibition. METHODS: Normal human enthesis soft tissue (ST) and adjacent perientheseal bone (PEB) (n=15) were evaluated using immunohistochemistry (IHC), digested for myeloid cell phenotyping, sorted and stimulated with different adjuvants (lipopolysaccharide and mannan). Stimulated enthesis fractions were analysed for inducible production of spondyloarthropathy disease-relevant mediators (IL-23 full protein, TNF, IL-1ß and CCL20). Myeloid populations were also compared with matched blood populations for further mRNA analysis and the effect of PDE4 inhibition was assessed. RESULTS: A myeloid cell population (CD45+ HLADR+ CD14+ CD11c+) phenotype was isolated from both the ST and adjacent PEB and termed 'CD14+ myeloid cells' with tissue localisation confirmed by CD14+ IHC. The CD14- fraction contained a CD123+ HLADR+ CD11c- cell population (plasmacytoid dendritic cells). The CD14+ population was the dominant entheseal producer of IL-23, IL-1ß, TNF and CCL20. IL-23 and TNF from the CD14+ population could be downregulated by a PDE4I and other agents (histamine and 8-Bromo-cAMP) which elevate cAMP. Entheseal CD14+ cells had a broadly similar gene expression profile to the corresponding CD14+ population from matched blood but showed significantly lower CCR2 gene expression. CONCLUSIONS: The human enthesis contains a CD14+ myeloid population that produces most of the inducible IL-23, IL-1ß, TNF and CCL20. This population has similar gene expression profile to the matched blood CD14+ population.

Spondyloarthritis: new insights into clinical aspects, translational immunology and therapeutics.

PURPOSE OF REVIEW: The spondyloarthopathies (SpA), which encompass related diseases that were originally viewed as autoimmune, are now known to have a strong innate immune or autoinflammatory initiation phase characterized by disease localization to tissue-specific sites based on the nuances and microanatomy and immunology of those sites. This review covers recent translational advances in the field of SpA. RECENT FINDINGS: Imaging studies in SpA continue to add support for the pivotal role of enthesitis in disease initiation and expression. Although in its infancy, there is growing evidence for microbiotal intestinal dysbiosis in ankylosing spondylitis and psoriatic arthritis. The role of cytokines beyond tumour necrosis factor (TNF) continues to grow with support for the interleukin (IL)-23/17 axis being key to disease and emergent evidence for the importance of the IL-36 pathway. The treatment of inflammatory bowel disease (IBD) with vedolizumab an α4ß7-integrin blocker has been associated with arthritis flares and small molecules with Janus kinase inhibition appear to be as effective as the anti-TNFs. The disparate response of different domains in SpA points towards immunological heterogeneity even within what was considered a homogeneous disease. SUMMARY: The clinical aspects and translational immunology and therapeutics of SpA continue to evolve and indicate the complexity of diagnosis and treatment of these conditions.

Enthesitis: Much More Than Focal Insertion Point Inflammation.

PURPOSE OF REVIEW: Recognition of the importance of enthesitis as the pivotal pathological process underpinning spondyloarthropathies (SpA) has increased in recent years. Thus, we summarized the current knowledge on the pathogenic role of enthesitis on SpA shown by both animal models and human studies in vivo. RECENT FINDINGS: Experimental models have shown several SpA-like diseases that commence at entheses and are linked to nail disease as well as dactylitis, two important entheseal-associated conditions in humans. Frequently, enthesitis is not the primary outcome measure in studies of peripheral PsA and SpA although arguably it is the key parameter being indirectly assessed in spinal disease in ankylosing spondylitis. The use of different agents including JAK, IL-17, and IL-23 inhibitors contributes significantly to our understanding of enthesitis in terms of involved immune pathways. Enthesitis and enthesis organ inflammation may be the primary pathological process underlying SpA associated skeletal inflammation. Emergent studies are beginning to elucidate the molecular basis for this type of joint inflammatory response.

High-density genetic mapping of a major QTL for resistance to multiple races of loose smut in a tetraploid wheat cross.

Loose smut, caused by Ustilago tritici (Pers.) Rostr., is a systemic disease of tetraploid durum wheat (Triticum turgidum L.). Loose smut can be economically controlled by growing resistant varieties, making it important to find and deploy new sources of resistance. Blackbird, a variety of T. turgidum L. subsp. carthlicum (Nevski) A. Love & D. Love, carries a high level of resistance to loose smut. Blackbird was crossed with the loose smut susceptible durum cultivar Strongfield to produce a doubled haploid (DH) mapping population. The parents and progenies were inoculated with U. tritici races T26, T32 and T33 individually and as a mixture at Swift Current, Canada in 2011 and 2012 and loose smut incidence (LSI) was assessed. Genotyping of the DH population and parents using an Infinium iSelect 90K single nucleotide polymorphism (SNP) array identified 12,952 polymorphic SNPs. The SNPs and 426 SSRs (previously genotyped in the same population) were mapped to 16 linkage groups spanning 3008.4 cM at an average inter-marker space of 0.2 cM in a high-density genetic map. Composite interval mapping analysis revealed three significant quantitative trait loci (QTL) for loose smut resistance on chromosomes 3A, 6B and 7A. The loose smut resistance QTL on 6B (QUt.spa-6B.2) and 7A (QUt.spa-7A.2) were derived from Blackbird. Strongfield contributed the minor QTL on 3A (QUt.spa-3A.2). The resistance on 6B was a stable major QTL effective against all individual races and the mixture of the three races; it explained up to 74% of the phenotypic variation. This study is the first attempt in durum wheat to identify and map loose smut resistance QTL using a high-density genetic map. The QTL QUt.spa-6B.2 would be an effective source for breeding resistance to multiple races of the loose smut pathogen because it provides near-complete broad resistance to the predominant virulence on the Canadian prairies.

The CD45lowCD271high Cell Prevalence in Bone Marrow Samples May Provide a Useful Measurement of the Bone Marrow Quality for Cartilage and Bone Regenerative Therapy.

BACKGROUND: Bone marrow aspirates and concentrates are increasingly being used for musculoskeletal regenerative therapies, providing bone and cartilage progenitors. However, the quality of these bone marrow samples remains imprecise within clinical settings. As there is a need for the development of these therapies, a method of counting CD45CD271 cells was optimized and tested as an indicator of bone marrow sample quality. METHODS: Bone marrow aspirates were collected from 54 donors (28 male and 26 female; median age of 48 years). The reagent concentrations were optimized for fast staining, and an acoustic-focusing flow cytometer (Attune) was used to enable automated CD45CD271 cell counting in bone marrow aspirates, bone marrow concentrates, and samples loaded onto a collagen scaffold. The CD45CD271 cell counts were compared with those obtained using another flow-cytometry-based method (LSR II) and with connective tissue progenitor (CTP) counts quantified using a colony forming unit-fibroblast (CFU-F) assay. RESULTS: The optimized method enabled the counting of CD45CD271 cells within only 15 minutes. The quantified cell counts (median, 1,520; range, 96 to 20,992 cells/mL of bone marrow) were positively correlated with the CTP counts (p < 0.0001; r = 0.7237). In agreement with CFU-F and LSR II-based assays, the CD45CD271 cell counts quantified using the Attune-based method decreased with age in the samples from female but not male donors (p = 0.0015 and p = 0.3877, respectively). A significant increase in CD45CD271 cell counts was detected following bone marrow concentration (mean, 5-fold; 95% confidence interval [CI], 3.6 to 7.2-fold). Additionally, the number of CD45CD271 cells attached to the collagen scaffold was positively correlated with the number of progenitor cells that survived on the scaffold after 2-week culture (p = 0.0348). CONCLUSIONS: An assay for counting CD45CD271 cells may provide a useful measurement of bone marrow quality. While the specificity of this measurement of CD45CD271 cells remained low in our experimental conditions, CD45CD271 cell counts were positively and modestly correlated with the prevalence of CTPs. CLINICAL RELEVANCE: A fast and automated assessment of bone marrow aspirate/concentrate quality using CD45CD271 cell counting may be a useful tool for improving the quality of regenerative therapy.

Paracrine cyclooxygenase-2 activity by macrophages drives colorectal adenoma progression in the Apc Min/+ mouse model of intestinal tumorigenesis.

Genetic deletion or pharmacological inhibition of cyclooxygenase (COX)-2 abrogates intestinal adenoma development at early stages of colorectal carcinogenesis. COX-2 is localised to stromal cells (predominantly macrophages) in human and mouse intestinal adenomas. Therefore, we tested the hypothesis that paracrine Cox-2-mediated signalling from macrophages drives adenoma growth and progression in vivo in the Apc Min/+ mouse model of intestinal tumorigenesis. Using a transgenic C57Bl/6 mouse model of Cox-2 over-expression driven by the chicken lysozyme locus (cLys-Cox-2), which directs integration site-independent, copy number-dependent transgene expression restricted to macrophages, we demonstrated that stromal macrophage Cox-2 in colorectal (but not small intestinal) adenomas from cLys-Cox-2 x Apc Min/+ mice was associated with significantly increased tumour size (P = 0.025) and multiplicity (P = 0.025), compared with control Apc Min/+ mice. Transgenic macrophage Cox-2 expression was associated with increased dysplasia, epithelial cell Cox-2 expression and submucosal tumour invasion, as well as increased nuclear ß-catenin translocation in dysplastic epithelial cells. In vitro studies confirmed that paracrine macrophage Cox-2 signalling drives catenin-related transcription in intestinal epithelial cells. Paracrine macrophage Cox-2 activity drives growth and progression of Apc Min/+ mouse colonic adenomas, linked to increased epithelial cell ß-catenin dysregulation. Stromal cell (macrophage) gene regulation and signalling represent valid targets for chemoprevention of colorectal cancer.

Interleukin-22 drives the proliferation, migration and osteogenic differentiation of mesenchymal stem cells: a novel cytokine that could contribute to new bone formation in spondyloarthropathies.

OBJECTIVES.: The SpAs are genetically and therapeutically linked to IL-23, which in turn regulates IL-22, a cytokine that has been implicated in the regulation of new bone formation in experimental models. We hypothesize that IL-22, a master regulator of stem cells in other niches, might also regulate human mesenchymal stem cell (MSC) osteogenesis. METHODS.: The effects of IL-22 on in vitro MSC proliferation, migration and osteogenic differentiation were evaluated in the presence or absence of IFN-γ and TNF (to ascertain IL-22 activity in pro-inflammatory environments). Colorimetric XTT assay, trans-well migration assays, quantitative real-time PCR (qRT-PCR) for MSC lineage markers and osteogenesis assays were used. RESULTS.: Combined treatment of MSC with IL-22, IFN-γ and TNF resulted in increased MSC proliferation ( P = 0.008) and migration ( P = 0.04), an effect that was not seen in cells treated with IL-22 alone and untreated cells. Osteogenic and adipogenic, but not chondrogenic, transcription factors were upregulated by IL-22 alone ( P < 0.05). MSC osteogenesis was enhanced following IL-22 exposure ( P = 0.03, measured by calcium production). The combination of IFN-γ and TNF with or without IL-22 suppressed MSC osteogenesis ( P = 0.03). CONCLUSION.: This work shows that IL-22 is involved in human MSC proliferation/migration in inflammatory environments, with MSC osteogenesis occurring only in the absence of IFN-γ/TNF. These effects of IL-22 on MSC function is a novel pathway for exploring pathological, post-inflammation osteogenesis in human SpA.

Examining the feasibility of clinical grade CD271+ enrichment of mesenchymal stromal cells for bone regeneration.

INTRODUCTION: Current clinical trials utilize mesenchymal stromal cells (MSCs) expanded in culture, however these interventions carry considerable costs and concerns pertaining to culture-induced losses of potency. This study assessed the feasibility of new clinical-grade technology to obtain uncultured MSC isolates from three human intra-osseous tissue sources based on immunomagnetic selection for CD271-positive cells. MATERIALS AND METHODS: MSCs were isolated from bone marrow (BM) aspirates or surgical waste materials; enzymatically digested femoral heads (FHs) and reamer irrigator aspirator (RIA) waste fluids. Flow cytometry for the CD45-/lowCD73+CD271+ phenotype was used to evaluate uncultured MSCs before and after selection, and to measure MSC enrichment in parallel to colony forming-unit fibroblast assay. Trilineage differentiation assays and quantitative polymerase chain-reaction for key transcripts involved in bone regeneration was used to assess the functional utility of isolated cells for bone repair. RESULTS: Uncultured CD45-/lowCD271+ MSCs uniformly expressed CD73, CD90 and CD105 but showed variable expression of MSCA-1 and SUSD2 (BM>RIA>FH). MSCs were enriched over 150-fold from BM aspirates and RIA fluids, whereas the highest MSC purities were obtained from FH digests. Enriched fractions expressed increased levels of BMP-2, COL1A2, VEGFC, SPARC and CXCL12 transcripts (BM>RIA>FH), with the highest up-regulation detected for CXCL12 in BM (>1300-fold). Following culture expansion, CD271-selected MSCS were tri-potential and phenotypically identical to plastic adherence-selected MSCs. DISCUSSION: A CD271-based GMP-compliant immunomagnetic selection resulted in a substantial increase in MSC purity and elevated expression of transcripts involved in bone formation, vascularisation and chemo-attraction. Although this technology, particularly from RIA fluids, can be immediately applied by orthopaedic surgeons as autologous therapy, further improvements in MSC purities and pre-clinical testing of product safety would be required to develop this process for allogeneic applications.

Multipotential stromal cell abundance in cellular bone allograft: comparison with fresh age-matched iliac crest bone and bone marrow aspirate.

AIM: To enumerate and characterize multipotential stromal cells (MSCs) in a cellular bone allograft and compare with fresh age-matched iliac crest bone and bone marrow (BM) aspirate. MATERIALS & METHODS: MSC characterization used functional assays, confocal/scanning electron microscopy and whole-genome microarrays. Resident MSCs were enumerated by flow cytometry following enzymatic extraction. RESULTS: Allograft material contained live osteocytes and proliferative bone-lining cells defined as MSCs by phenotypic and functional capacities. Without cultivation/expansion, the allograft displayed an 'osteoinductive' molecular signature and the presence of CD45(-)CD271(+)CD73(+)CD90(+)CD105(+) MSCs; with a purity over 100-fold that of iliac crest bone. In comparison with BM, MSC numbers enzymatically released from 1 g of cellular allograft were equivalent to approximately 45 ml of BM aspirate. CONCLUSION: Cellular allograft bone represents a unique nonimmune material rich in MSCs and osteocytes. This osteoinductive graft represents an attractive alternative to autograft bone or composite/synthetic grafts in orthopedics and broader regenerative medicine settings.

Induced periosteum a complex cellular scaffold for the treatment of large bone defects.

OBJECTIVE: Surgically induced periosteal membrane holds great potential for the treatment of large bone defects representing a simple alternative to combinations of exogenous stem cells, scaffolds and growth factors. The purpose of this study was to explore the biological basis for this novel regenerative medicine strategy in man. METHODS: Eight patients with critical size defects were treated with the induced membrane (IM) technique. After membrane formation 1cm(2) biopsy was taken together with matched, healthy diaphyseal periosteum (P) for comparative analysis. Morphological characteristics, cell composition and growth factor expression were compared. Functional and molecular evaluation of mesenchymal stromal cell (MSC) activity was performed. RESULTS: Both tissues shared similar morphology although IM was significantly thicker than P (p=0.032). The frequency of lymphocytes, pericytes (CD45(-)CD34(-)CD146(+)) and cells expressing markers consistent with bone marrow MSCs (CD45(-/low)CD271(bright)) were 31. 3 and 15.5-fold higher respectively in IM (all p=0.043). IM contained 3-fold more cells per gramme of tissue with a similar proportion of endothelial cells (CD45(-)CD31(+)). Expressed bone morphogenic protein 2, vascular endothelial growth factor and stromal derived factor 1 (SDF-1) are key tissue regeneration mediators. Adherent expanded cells from both tissues had molecular profiles similar to bone marrow MSCs but cells from IM expressed greater than 2 fold relative abundance of SDF-1transcript compared to P (p=0.043). CONCLUSION: The IM is a thick, vascularised structure that resembles periosteum with a cellular composition and molecular profile facilitating large defect repair and therefore may be described as an "induced-periosteum". This tissue offers a powerful example of in situ 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.

Single-platform quality control assay to quantify multipotential stromal cells in bone marrow aspirates prior to bulk manufacture or direct therapeutic use.

BACKGROUND AIMS: The manufacture of multipotential stromal cell (MSC)-based products is costly; therefore, a rapid evaluation of bone marrow (BM) 'quality' with respect to MSC content is desirable. The aim of this study was to develop a rapid single-platform assay to quantify MSC in BM aspirates. METHODS: Aspirated MSC were enumerated using the CD45(-/low) CD271(bright) phenotype and AccuCheck counting beads and compared with a classic colony-forming unit-fibroblast (CFU-F) assay. The phenotype of CD45(-/low) CD271(bright) cells was defined using a range of MSC (CD73, CD105, CD90) and non-MSC (CD31, CD33, CD34, CD19) markers. The effect of aspirated BM volume on MSC yield was also determined. RESULTS: CD45(-/low) CD271(bright) cells had a classic MSC phenotype (CD73(+) CD105(+) CD90(+)). Their numbers correlated positively with CFU-F counted manually (R = 0.81, P < 0.001) or using automatic measurements of surface area occupied by colonies (R = 0.66, P < 0.001). Simultaneous enumeration of CD34(+) cells revealed donor variability ranges compatible with standard International Society of Hematotherapy and Graft Engineering (ISHGE) protocols. Aspirating larger marrow volumes gave a significant several-fold reduction in the frequency of CFU-F and CD45(-/low) CD271(bright) cells per milliliter. Therefore aspirated MSC yields can be maximized through a standardized, low-volume harvesting technique. CONCLUSIONS: Absolute quantification of CD45(-/low) CD271(bright) cells was found to be a reliable method of predicting CFU-F yields in BM aspirates. This rapid (< 40 min) procedure could be suitable for intra-operative quality control of BM aspirates prior to volume reduction/direct injection in orthopedics. In the production of culture-expanded MSC, this assay could be used to exclude samples containing low numbers of MSC, resulting in improved consistency and quality of manufactured MSC batches.