Non-destructive, label free identification of cell cycle phase in cancer cells by multispectral microscopy of autofluorescence.

BACKGROUND: Cell cycle analysis is important for cancer research. However, available methodologies have drawbacks including limited categorisation and reliance on fixation, staining or transformation. Multispectral analysis of endogenous cell autofluorescence has been shown to be sensitive to changes in cell status and could be applied to the discrimination of cell cycle without these steps. METHODS: Cells from the MIA-PaCa-2, PANC-1, and HeLa cell lines were plated on gridded dishes and imaged using a multispectral fluorescence microscope. They were then stained for proliferating cell nuclear antigen (PCNA) and DNA intensity as a reference standard for their cell cycle position (G1, S, G2, M). The multispectral data was split into training and testing datasets and models were generated to discriminate between G1, S, and G2 + M phase cells. A standard decision tree classification approach was taken, and a two-step system was generated for each line. RESULTS: Across cancer cell lines accuracy ranged from 68.3% (MIA-PaCa-2) to 73.3% (HeLa) for distinguishing G1 from S and G2 + M, and 69.0% (MIA-PaCa-2) to 78.0% (PANC1) for distinguishing S from G2 + M. Unmixing the multispectral data showed that the autofluorophores NADH, FAD, and PPIX had significant differences between phases. Similarly, the redox ratio and the ratio of protein bound to free NADH were significantly affected. CONCLUSIONS: These results demonstrate that multispectral microscopy could be used for the non-destructive, label free discrimination of cell cycle phase in cancer cells. They provide novel information on the mechanisms of cell-cycle progression and control, and have practical implications for oncology research.

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.

Incremental benefits of repeated mesenchymal stromal cell administration compared with solitary intervention after myocardial infarction.

BACKGROUND AIMS: Traditionally, stem cell therapy for myocardial infarction (MI) has been administered as a single treatment in the acute or subacute period after MI. These time intervals coincide with marked differences in the post-infarct myocardial environment, raising the prospect that repeat cell dosing could provide incremental benefit beyond a solitary intervention. This prospect was evaluated with the use of mesenchymal stromal cells (MSCs). METHODS: Three groups of rats were studied. Single-therapy and dual-therapy groups received allogeneic, prospectively isolated MSCs (1 × 10(6) cells) by trans-epicardial injection immediately after MI, with additional dosing 1 week later in the dual-therapy cohort. Control animals received cryopreservant solution only. Left ventricular (LV) dimensions and ejection fraction (EF) were assessed by cardiac magnetic resonance immediately before MI and at 1, 2 and 4 weeks after MI. RESULTS: Immediate MSC treatment attenuated early myocardial damage with EF of 35.3 ± 3.1% (dual group, n = 12) and 35.2 ± 2.2% (single group, n = 15) at 1 week after MI compared with 22.1 ± 1.9% in controls (n = 17, P < 0.01). In animals receiving a second dose of MSCs, EF increased to 40.7 ± 3.1% by week 4, which was significantly higher than in the single-therapy group (EF 35.9 ± 1.8%, P < 0.05). Dual MSC treatment was also associated with greater myocardial mass and arteriolar density, with trends toward reduced myocardial fibrosis. These incremental benefits were especially observed in remote (non-infarct) segments of LV myocardium. CONCLUSIONS: Repeated stem cell intervention in both the acute and the sub-acute period after MI provides additional improvement in ventricular function beyond solitary cell dosing, largely owing to beneficial changes remote to the area of infarction.

Impact of timing and dose of mesenchymal stromal cell therapy in a preclinical model of acute myocardial infarction.

BACKGROUND: Although mesenchymal stem/stromal cells (MSC) have shown therapeutic promise after myocardial infarction (MI), the impact of cell dose and timing of intervention remains uncertain. We compared immediate and deferred administration of 2 doses of MSC in a rat model of MI. METHODS AND RESULTS: Sprague-Dawley rats were used. Allogeneic prospectively isolated MSC ("low" dose 1 × 10(6) or "high" dose 2 × 10(6) cells) were delivered by transepicardial injection immediately after MI ("early-low," "early-high"), or 1 week later ("late-low," "late-high"). Control subjects received cryopreservant solution alone. Left ventricular dimensions and ejection fraction (EF) were assessed by cardiac magnetic resonance. All 4 MSC-treatment cohorts demonstrated higher EF than control animals 4 weeks after MI (P values <.01 to <.0001), with function most preserved in the early-high group (absolute reduction in EF from baseline: control 39.1 ± 1.7%, early-low 26.5 ± 3.2%, early-high 7.9 ± 2.6%, late-low 19.6 ± 3.5%, late-high 17.9 ± 4.0%). Cell treatment also attenuated left ventricular dilatation and fibrosis and augmented left ventricular mass, systolic wall thickening (SWT), and microvascular density. Although early intervention selectively increased SWT and vascular density in the infarct territory, delayed treatment caused greater benefit in remote (noninfarct) myocardium. All outcomes demonstrated dose dependence for early MSC treatment, but not for later cell administration. CONCLUSIONS: The nature and magnitude of benefit from MSC after acute MI is strongly influenced by timing of cell delivery, with dose dependence most evident for early intervention. These novel insights have potential implications for cell therapy after MI in human patients.

A senescence stress secretome is a hallmark of therapy-related myeloid neoplasm stromal tissue occurring soon after cytotoxic exposure.

Therapy-related myeloid neoplasm (tMN) is considered a direct consequence of DNA damage in hematopoietic stem cells. Despite increasing recognition that altered stroma can also drive leukemogenesis, the functional biology of the tMN microenvironment remains unknown. We performed multiomic (transcriptome, DNA damage response, cytokine secretome and functional profiling) characterization of bone marrow stromal cells from tMN patients. Critically, we also compared (i) patients with myeloid neoplasm and another cancer but without cytotoxic exposure, (ii) typical primary myeloid neoplasm, and (iii) age-matched controls to decipher the microenvironmental changes induced by cytotoxics vs. neoplasia. Strikingly, tMN exhibited a profoundly senescent phenotype with induction of CDKN1A and ß-Galactosidase, defective phenotype, and proliferation. Moreover, tMN stroma showed delayed DNA repair and defective adipogenesis. Despite their dormant state, tMN stromal cells were metabolically highly active with a switch toward glycolysis and secreted multiple pro-inflammatory cytokines indicative of a senescent-secretory phenotype that inhibited adipogenesis. Critically, senolytics not only eliminated dormant cells, but also restored adipogenesis. Finally, sequential patient sampling showed senescence phenotypes are induced within months of cytotoxic exposure, well prior to the onset of secondary cancer. Our data underscores a role of senescence in the pathogenesis of tMN and provide a valuable resource for future therapeutics.

Ageing human bone marrow mesenchymal stem cells have depleted NAD(P)H and distinct multispectral autofluorescence.

Stem cell exhaustion plays a major role in the ageing of different tissues. Similarly, in vitro cell ageing during expansion prior to their use in regenerative medicine can severely compromise stem cell quality through progressive declines in differentiation and growth capacity. We utilized non-destructive multispectral assessment of native cell autofluorescence to investigate the metabolic mechanisms of in vitro mesenchymal stem cell (MSC) ageing in human bone marrow MSCs over serial passages (P2-P10). The spectral signals for NAD(P)H, flavins and protein-bound NAD(P)H were successfully isolated using Robust Dependent Component Analysis (RoDECA). NAD(P)H decreased over the course of hMSC ageing in absolute terms as well as relative to flavins (optical redox ratio). Relative changes in other fluorophore levels (flavins, protein-bound NAD(P)H) suggested that this reduction was due to nicotinamide adenine dinucleotide depletion rather than a metabolic shift from glycolysis to oxidative phosphorylation. Using multispectral features, which are determined without cell fixation or fluorescent labelling, we developed and externally validated a reliable, linear model which could accurately categorize the age of culture-expanded hMSCs. The largest shift in spectral characteristics occurs early in hMSC ageing. These findings demonstrate the feasibility of applying multispectral technology for the non-invasive monitoring of MSC health in vitro.

Conditional knockout of ephrinB1 in osteogenic progenitors delays the process of endochondral ossification during fracture repair.

The Eph receptor tyrosine kinase ligand, ephrinB1 (EfnB1) is important for correct skeletal and cartilage development, however, the role of EfnB1 in fracture repair is unknown. This study investigated the role of EfnB1 during fracture repair where EfnB1 expression increased significantly at 1 and 2 weeks post fracture in C57Bl/6 wildtype mice, coinciding with the haematoma, soft callus formation/remodelling stages, respectively. To investigate the specific role of EfnB1 within the osteogenic lineage during fracture repair, male mice with a conditional deletion of EfnB1 in the osteogenic lineage (EfnB1OBfl/O), driven by the Osterix (Osx) promoter, and their male Osx:Cre counterparts were subject to a femoral fracture with internal fixation. Two weeks post fracture micro computed tomography (µCT) analysis revealed that EfnB1OBfl/O mice displayed a significant decrease in bone volume relative to tissue volume within the fracture callus. This was attributed to an alteration in the distribution of osteoclasts within the fracture site, a significant elevation in cartilaginous tissue and reduction in the osteoprogenitor population and calcein labelled bone within the fracture site of EfnB1OBfl/O mice. Supportive in vitro studies demonstrated that under osteogenic conditions, cultured EfnB1OBfl/O stromal cells derived from the 2 week fracture site exhibited a reduced capacity to produce mineral and decreased expression of the osteogenic gene, Osterix, when compared to Osx:Cre controls. These findings suggest that the loss of EfnB1 delays the fracture repair process. The present study confirmed that EFNB1 activation in human BMSC, following stimulation with soluble-EphB2 resulted in de-phosphorylation of TAZ, demonstrating similarities in EfnB1 signalling between human and mouse stromal populations. Overall, the present study provides evidence that loss of EfnB1 in the osteo/chondrogenic lineages delays the soft callus formation/remodelling stages of the fracture repair process.

Loss of EfnB1 in the osteogenic lineage compromises their capacity to support hematopoietic 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.

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.

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-1(bright) 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.

Pentosan polysulfate binds to STRO-1+ mesenchymal progenitor cells, is internalized, and modifies gene expression: a novel approach of pre-programing stem cells for therapeutic application requiring their chondrogenesis.

BACKGROUND: The pharmaceutical agent pentosan polysulfate (PPS) is known to induce proliferation and chondrogenesis of mesenchymal progenitor cells (MPCs) in vitro and in vivo. However, the mechanism(s) of action of PPS in mediating these effects remains unresolved. In the present report we address this issue by investigating the binding and uptake of PPS by MPCs and monitoring gene expression and proteoglycan biosynthesis before and after the cells had been exposed to limited concentrations of PPS and then re-established in culture in the absence of the drug (MPC priming). METHODS: Immuno-selected STRO-1+ mesenchymal progenitor stem cells (MPCs) were prepared from human bone marrow aspirates and established in culture. The kinetics of uptake, shedding, and internalization of PPS by MPCs was determined by monitoring the concentration-dependent loss of PPS media concentrations using an enzyme-linked immunosorbent assay (ELISA) and the uptake of fluorescein isothiocyanate (FITC)-labelled PPS by MPCs. The proliferation of MPCs, following pre-incubation and removal of PPS (priming), was assessed using the Wst-8 assay method, and proteoglycan synthesis was determined by the incorporation of 35SO4 into their sulphated glycosaminoglycans. The changes in expression of MPC-related cell surface antigens of non-primed and PPS-primed MPCs from three donors was determined using flow cytometry. RNA sequencing of RNA isolated from non-primed and PPS-primed MPCs from the same donors was undertaken to identify the genes altered by the PPS priming protocol. RESULTS: The kinetic studies indicated that, in culture, PPS rapidly binds to MPC surface receptors, followed by internalisation and localization within the nucleus of the cells. Following PPS-priming of MPCs and a further 48 h of culture, both cell proliferation and proteoglycan synthesis were enhanced. Reduced expression of MPC-related cell surface antigen expression was promoted by the PPS priming, and RNA sequencing analysis revealed changes in the expression of 42 genes. CONCLUSION: This study has shown that priming of MPCs with low concentrations of PPS enhanced chondrogenesis and MPC proliferation by modifying their characteristic basal gene and protein expression. These findings offer a novel approach to re-programming mesenchymal stem cells for clinical indications which require the repair or regeneration of cartilaginous tissues such as in osteoarthritis and degenerative disc disease.

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.

Comparative assessment of the osteoconductive properties of different biomaterials in vivo seeded with human or ovine mesenchymal stem/stromal cells.

Mesenchymal stromal/stem cells (MSC), when used in combination with biomaterial scaffolds, have been shown to contribute at varying efficiencies to bone and cartilage regeneration in preclinical large animal models and human clinical trials. In an orthopedic context, identification of the optimal scaffold, which is capable of inducing tissue regeneration, has been the subject of numerous studies. In the present study, we show that ex vivo-expanded MSC from human and ovine bone marrow display similar phenotypic properties, but exhibit differences in their ability to form bone in vivo when transplanted with different biocompatible scaffold composites. We found that the ovine MSC formed ectopic bone on all scaffolds tested with the exception of collagen-based demineralized bone matrix. In contrast, human MSC in general formed less bone and only on those biomaterials composed of ceramic particles containing at least 15% hydroxyapatite. This study demonstrates the differences in bone formation potential between human and ovine MSC in vivo based on the osteoconductive properties of different bioscaffolds currently being used for orthopedic clinical applications.

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.