Sharon Paton

Multipotential human adipose-derived stromal stem cells exhibit a perivascular phenotype in vitro and in vivo.

Mesenchymal stem‐like cells identified in different tissues reside in a perivascular niche. In the present study, we investigated the putative niche of adipose‐derived stromal/stem cells (ASCs) using markers, associated with mesenchymal and perivascular cells, including STRO‐1, CD146, and 3G5. Immunofluorescence staining of human adipose tissue sections, revealed that STRO‐1 and 3G5 co‐localized with CD146 to the perivascular regions of blood vessels. FACS was used to determine the capacity of the CD146, 3G5, and STRO‐1 specific monoclonal antibodies to isolate clonogenic ASCs from disassociated human adipose tissue. Clonogenic fibroblastic colonies (CFU‐F) were found to be enriched in those cell fractions selected with either STRO‐1, CD146, or 3G5. Flow cytometric analysis revealed that cultured ASCs exhibited similar phenotypic profiles in relation to their expression of cell surface markers associated with stromal cells (CD44, CD90, CD105, CD106, CD146, CD166, STRO‐1, alkaline phosphatase), endothelial cells (CD31, CD105, CD106, CD146, CD166), haematopoietic cells (CD14, CD31, CD45), and perivascular cells (3G5, STRO‐1, CD146). The immunoselected ASCs populations maintained their characteristic multipotential properties as shown by their capacity to form Alizarin Red positive mineralized deposits, Oil Red O positive lipid droplets, and Alcian Blue positive proteoglycan‐rich matrix in vitro. Furthermore, ASCs cultures established from either STRO‐1, 3G5, or CD146 selected cell populations, were all capable of forming ectopic bone when transplanted subcutaneously into NOD/SCID mice. The findings presented here, describe a multipotential stem cell population within adult human adipose tissue, which appear to be intimately associated with perivascular cells surrounding the blood vessels. J. Cell. Physiol. 214: 413–421, 2008.

Enrichment for STRO-1 expression enhances the cardiovascular paracrine activity of human bone marrow-derived mesenchymal cell populations.

The cardiovascular therapeutic potential of bone marrow mesenchymal stromal/stem cells (MSC) is largely mediated by paracrine effects. Traditional preparation of MSC has involved plastic adherence‐isolation. In contrast, prospective immunoselection aims to improve cell isolation by enriching for mesenchymal precursor cells (MPC) at higher purity. This study compared the biological characteristics and cardiovascular trophic activity of plastic adherence‐isolated MSC (PA‐MSC) and MPC prepared from the same human donors by immunoselection for stromal precursor antigen‐1 (STRO‐1). Compared to PA‐MSC, STRO‐1‐MPC displayed greater (1) clonogenicity, (2) proliferative capacity, (3) multilineage differentiation potential, and (4) mRNA expression of mesenchymal stem cell‐related transcripts. In vitro assays demonstrated that conditioned medium from STRO‐1‐MPC had greater paracrine activity than PA‐MSC, with respect to cardiac cell proliferation and migration and endothelial cell migration and tube formation. In keeping with this, STRO‐1‐MPC exhibited higher gene and protein expression of CXCL12 and HGF. Inhibition of these cytokines attenuated endothelial tube formation and cardiac cell proliferation, respectively. Paracrine responses were enhanced by using supernatant from STRO‐1Bright MPC and diminished with STRO‐1Dim conditioned medium. Together, these findings indicate that prospective isolation gives rise to mesenchymal progeny that maintain a higher proportion of immature precursor cells compared to traditional plastic adherence‐isolation. Enrichment for STRO‐1 is also accompanied by increased expression of cardiovascular‐relevant cytokines and enhanced trophic activity. Immunoselection thus provides a strategy for improving the cardiovascular reparative potential of mesenchymal cells. J. Cell. Physiol. 223: 530–540, 2010.

Human mulipotential mesenchymal/stromal stem cells are derived from a discrete subpopulation of STRO-1bright/CD34−/CD45−/glycophorin-A-bone marrow cells

Magnetic and flow cytometry-based methods were used to characterize clonogenic stromal cells in human bone marrow. STRO-1bright stromal cells were found to lack expression of CD34, CD45 and glycophorin-A markers associated with hematopoietic progenitor cells. These studies support the view that these are two distinct stem cell compartments in adult bone marrow.

Heat Shock Protein-90 beta Is Expressed at the Surface of Multipotential Mesenchymal Precursor Cells: Generation of a Novel Monoclonal Antibody, STRO-4, With Specificity for Mesenchymal Precursor Cells From Human and Ovine Tissues

Mesenchymal stromal cells (MSCs) and their precursor cells (MPCs) can proliferate and differentiate into multiple mesodermal and some ectodermal and endodermal tissues. Culture-expanded MSCs are currently being evaluated as a possible cell therapy to replace/repair injured or diseased tissues. While a number of mAb reagents with specificity to human MSCs, including STRO-1, STRO-3 (BLK ALP), CD71 (SH2, SH3), CD106 (VCAM-1), CD166, and CD271, have facilitated the isolation of purified populations of human MSCs from primary tissues, few if any mAb reagents have been described that can be used to isolate equivalent cells from other species. This is of particular relevance when assessing the tissue regenerative efficacy of MSCs in large immunocompetent, preclinical animal models of disease. In light of this, we sought to generate novel monoclonal antibodies (mAb) with specific reactivity against a cell surface molecule that is expressed at high levels by MSCs from different species. Using CD106 (VCAM-1)-selected ovine MSCs as an immunogen, mAb-producing hybridomas were selected for their reactivity to both human and ovine MSCs. One such hybridoma, termed STRO-4, produced an IgG mAb that reacted with <5% of human and ovine bone marrow (BM) mononuclear cells. As a single selection reagent, STRO-4 mAb was able to enrich colony-forming fibroblasts (CFU-F) in both human and ovine BM by 16- and 8-folds, respectively. Cells isolated with STRO-4 exhibited reactivity with markers commonly associated with MSCs isolated by plastic adherence including CD29, CD44, and CD166. Moreover, when placed in inductive culture conditions in vitro, STRO-4(+) MSCs exhibited multilineage differentiation potential and were capable of forming a mineralized matrix, lipid-filled adipocytes, and chondrocytes capable of forming a glycosaminoglycan-rich matrix. Biochemical analysis revealed that STRO-4 identified the beta isoform of heat shock protein-90 (Hsp90beta). In addition to identifying an antibody reagent that identifies a highly conserved epitope expressed by MSCs from different species, our study also points to a potential role for Hsp90beta in MSC biology.

EphB4 expressing stromal cells exhibit an enhanced capacity for hematopoietic stem cell maintenance

The tyrosine kinase receptor, EphB4, mediates cross‐talk between stromal and hematopoietic populations during bone remodeling, fracture repair and arthritis, through its interactions with the ligand, ephrin‐B2. This study demonstrated that transgenic EphB4 mice (EphB4 Tg), over‐expressing EphB4 under the control of collagen type‐1 promoter, exhibited higher frequencies of osteogenic cells and hematopoietic stem/progenitor cells (HSC), correlating with a higher frequency of long‐term culture‐initiating cells (LTC‐IC), compared with wild type (WT) mice. EphB4 Tg stromal feeder layers displayed a greater capacity to support LTC‐IC in vitro, where blocking EphB4/ephrin‐B2 interactions decreased LTC‐IC output. Similarly, short hairpin RNA‐mediated EphB4 knockdown in human bone marrow stromal cells reduced their ability to support high ephrin‐B2 expressing CD34+ HSC in LTC‐IC cultures. Notably, irradiated EphB4 Tg mouse recipients displayed enhanced bone marrow reconstitution capacity and enhanced homing efficiency of transplanted donor hematopoietic stem/progenitor cells relative to WT controls. Studies examining the expression of hematopoietic supportive factors produced by stromal cells indicated that CXCL12, Angiopoietin‐1, IL‐6, FLT‐3 ligand, and osteopontin expression were more highly expressed in EphB4 Tg stromal cells compared with WT controls. These findings indicate that EphB4 facilitates stromal‐mediated support of hematopoiesis, and constitute a novel component of the HSC niche. Stem Cells 2015;33:2838—2849

Cardiac magnetic resonance, transthoracic and transoesophageal echocardiography: a comparison of in vivo assessment of ventricular function in rats

In vivo assessment of ventricular function in rodents has largely been restricted to transthoracic echocardiography (TTE). However 1.5 T cardiac magnetic resonance (CMR) and transoesophageal echocardiography (TOE) have emerged as possible alternatives. Yet, to date, no study has systematically assessed these three imaging modalities in determining ejection fraction (EF) in rats. Twenty rats underwent imaging four weeks after surgically-induced myocardial infarction. CMR was performed on a 1.5 T scanner, TTE was conducted using a 9.2 MHz transducer and TOE was performed with a 10 MHz intracardiac echo catheter. Correlation between the three techniques for EF determination and analysis reproducibility was assessed. Moderate-strong correlation was observed between the three modalities; the greatest between CMR and TOE (intraclass correlation coefficient (ICC) = 0.89), followed by TOE and TTE (ICC = 0.70) and CMR and TTE (ICC = 0.63). Intra- and inter-observer variations were excellent with CMR (ICC = 0.99 and 0.98 respectively), very good with TTE (0.90 and 0.89) and TOE (0.87 and 0.84). Each modality is a viable option for evaluating ventricular function in rats, however the high image quality and excellent reproducibility of CMR offers distinct advantages even at 1.5 T with conventional coils and software.

Loss of ephrinB1 in osteogenic progenitor cells impedes endochondral ossification and compromises bone strength integrity during skeletal development

The EphB receptor tyrosine kinase family and their ephrinB ligands have been implicated as mediators of skeletal development and bone homeostasis in humans, where mutations in ephrinB1 contribute to frontonasal dysplasia and coronal craniosynostosis. In mouse models, ephrinB1 has been shown to be a critical factor mediating osteoblast function. The present study examined the functional importance of ephrinB1 during endochondral ossification using the Cre recombination system with targeted deletion of ephrinB1 (EfnB1fl/fl) in osteogenic progenitor cells, under the control of the osterix (Osx:Cre) promoter. The Osx:EfnB1-/- mice displayed aberrant bone growth during embryonic and postnatal skeletal development up to 4weeks of age, when compared to the Osx:Cre controls. Furthermore, compared to the Osx:Cre control mice, the Osx:EfnB1-/- mice exhibited significantly weaker and less rigid bones, with a reduction in trabecular/ cortical bone formation, reduced trabecular architecture and a reduction in the size of the growth plates at the distal end of the femora from newborn through to 4weeks of age. The aberrant bone formation correlated with increased numbers of tartrate resistant acid phosphatase positive osteoclasts and decreased numbers of bone lining osteoblasts in 4week old Osx:EfnB1-/- mice, compared to Osx:Cre control mice. Taken together, these observations demonstrate the importance of ephrinB1 signalling between cells of the skeleton required for endochondral ossification.

The osteoprogenitor-specific loss of ephrinB1 results in an osteoporotic phenotype affecting the balance between bone formation and resorption

The present study investigated the effects of conditional deletion of ephrinB1 in osteoprogenitor cells driven by the Osterix (Osx) promoter, on skeletal integrity in a murine model of ovariectomy-induced (OVX) osteoporosis. Histomorphometric and μCT analyses revealed that loss of ephrinB1 in sham Osx:cre-ephrinB1fl/fl mice caused a reduction in trabecular bone comparable to OVX Osx:Cre mice, which was associated with a significant reduction in bone formation rates and decrease in osteoblast numbers. Interestingly, these observations were not exacerbated in OVX Osx:cre-ephrinB1fl/fl mice. Furthermore, sham Osx:cre-ephrinB1fl/fl mice displayed significantly higher osteoclast numbers and circulating degraded collagen type 1 compared to OVX Osx:Cre mice. Confirmation studies found that cultured monocytes expressing EphB2 formed fewer TRAP+ multinucleated osteoclasts and exhibited lower resorption activity in the presence of soluble ephrinB1-Fc compared to IgG control. This inhibition of osteoclast formation and function induced by ephrinB1-Fc was reversed in the presence of an EphB2 chemical inhibitor. Collectively, these observations suggest that ephrinB1, expressed by osteoprogenitors, influences bone loss during the development of osteoporosis, by regulating both osteoblast and osteoclast formation and function, leading to a loss of skeletal integrity.

Loss of EfnB1 in the osteogenic lineage compromises their capacity to support haematopoietic stem/ progenitor cell maintenance

The bone marrow stromal microenvironment contributes to the maintenance and function of hematopoietic stem/progenitor cells (HSPCs). The Eph receptor tyrosine kinase family members have been implicated in bone homeostasis and stromal support of HSPCs. The present study examined the influence of EfnB1-expressing osteogenic lineage on HSPC function. Mice with conditional deletion of EfnB1 in the osteogenic lineage (EfnB1OB-/-), driven by the Osterix promoter, exhibited a reduced prevalence of osteogenic progenitors and osteoblasts, correlating to lower numbers of HSPCs compared with Osx:Cre mice. Long-term culture-initiating cell (LTC-IC) assays confirmed that the loss of EfnB1 within bone cells hindered HSPC function, with a significant reduction in colony formation in EfnB1OB-/- mice compared with Osx:Cre mice. Human studies confirmed that activation of EPHB2 on CD34+ HSPCs via EFNB1-Fc stimulation enhanced myeloid/erythroid colony formation, whereas functional blocking of either EPHB1 or EPHB2 inhibited the maintenance of LTC-ICs. Moreover, EFNB1 reverse signaling in human and mouse stromal cells was found to be required for the activation of the HSPC-promoting factor CXCL12. Collectively, the results of this study confirm that EfnB1 contributes to the stromal support of HSPC function and maintenance and may be an important factor in regulating the HSPC niche.