Establishing criteria for human mesenchymal stem cell potency

This study sought to identify critical determinants of mesenchymal stem cell (MSC) potency using in vitro and in vivo attributes of cells isolated from the bone marrow of age‐ and sex‐matched donors. Adherence to plastic was not indicative of potency, yet capacity for long‐term expansion in vitro varied considerably between donors, allowing the grouping of MSCs from the donors into either those with high‐growth capacity or low‐growth capacity. Using this grouping strategy, high‐growth capacity MSCs were smaller in size, had greater colony‐forming efficiency, and had longer telomeres. Cell‐surface biomarker analysis revealed that the International Society for Cellular Therapy (ISCT) criteria did not distinguish between high‐growth capacity and low‐growth capacity MSCs, whereas STRO‐1 and platelet‐derived growth factor receptor alpha were preferentially expressed on high‐growth capacity MSCs. These cells also had the highest mean expression of the mRNA transcripts TWIST‐1 and DERMO‐1. Irrespective of these differences, both groups of donor MSCs produced similar levels of key growth factors and cytokines involved in tissue regeneration and were capable of multilineage differentiation. However, high‐growth capacity MSCs produced approximately double the volume of mineralized tissue compared to low‐growth capacity MSCs when assessed for ectopic bone‐forming ability. The additional phenotypic criteria presented in this study when combined with the existing ISCT minimum criteria and working proposal will permit an improved assessment of MSC potency and provide a basis for establishing the quality of MSCs prior to their therapeutic application

Bovine Colostrum Supplementation and Bone Health: a Pilot Study

Research has shown the positive effects of some bovine colostrum components in bone cells; for instance, lactoferrin is reported to stimulate osteoblast proliferation and inhibit osteoclast activity in cell cultures. However, whether bovine colostrum as a whole can induce bone mass gains in osteoporotic bones is relatively unclear. The aim of this study was to investigate the effects of bovine colostrum supplementation in ovariectomized-induced bone loss (OVX) rats. Methods: Twenty-seven-month-old female Wister rats (n=16) were randomly assigned to the following two groups: 1) a healthy control (non-OVX) with no supplementation, and 2) a OVX with bovine colostrum supplementation (0.5g/day; oral consumption). After 5 months supplementation, bone microstructure was scanned using micro-CT (right tibia). Bone formation markers (serum: pre-and post supplementation) were analysed (alkaline phosphatase and osteocalcin) by ECLIA. The study was approved by the National Ethics Committee for the Use of Animals in Research (ORBEA). Results: No significant differences were found between groups in serum alkaline phosphatase either before or after supplementation (p>0.05). Serum osteocalcin significantly increased post-supple-mentation in the OVX compared to pre-supplementation (pre: 11.32+/-1.61; post: 12.45+/-1.21μg/L, p0.05). Trabecular bone mineral content (BMC), trabecular thickness, cortical bone mineral density (BMD) and cortical BMC were similar between groups after supplementation (p>0.05). However, OVX group revealed significantly higher trabecular porosity (5.6%, p<0.01), trabecular separation (36.3%, p<0.01), and cortical porosity (8.0%, p<0.01) compared to the healthy control post-supplementation. Conclusion: Bovine colostrum seems to preserve bone mass of OVX by stimulating bone formation. However, these positive effects seem not to be sufficient to restore bone micro-architecture in the OVX group, possibly because the administrated dose of bovine colostrum was not sufficient for OVX to catch-up healthy rats in terms of trabecular and cortical porosity. The potential therapeutic use of bovine colostrum for osteoporosis deserves further investigation

Enhancing the efficacy of stem cell therapy with glycosaminoglycans

Human mesenchymal stem cell (hMSC) therapy offers significant potential for osteochondral regeneration. Such applications require their ex vivo expansion in media frequently supplemented with fibroblast growth factor 2 (FGF2). Particular heparan sulfate (HS) fractions stabilize FGF2-FGF receptor complexes. We show that an FGF2-binding HS variant (HS8) accelerates the expansion of freshly isolated bone marrow hMSCs without compromising their naivety. Importantly, the repair of osteochondral defects in both rats and pigs is improved after treatment with HS8-supplemented hMSCs (MSCHS8), when assessed histologically, biomechanically, or by MRI. Thus, supplementing hMSC culture media with an HS variant that targets endogenously produced FGF2 allows the elimination of exogenous growth factors that may adversely affect their therapeutic potency

An unbiased proteomics approach to identify the senescence-associated secretory phenotype of human bone marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) derived from bone marrow can support skeletal tissue repair and regeneration owing to their self-renewing capacity, differentiation ability, and trophic functions. Bone marrow-derived MSCs undergo dramatic changes with aging, including the senescence-associated secretory phenotype (SASP) which may largely contribute to age-related changes in bone tissue leading to osteoporosis. A mass spectrometry-based proteomics approach was used to investigate the MSC SASP. Replicative senescence was achieved by exhaustive in vitro sub-cultivation and confirmed by standard proliferation criteria. Conditioned media from non-senescent and senescent MSCs underwent mass spectrometry. Proteomics and bioinformatics analyses enabled the identification of 95 proteins expressed uniquely in senescent MSCs. Protein ontology analysis revealed the enrichment of proteins linked to the extracellular matrix, exosomes, cell adhesion, and calcium ion binding. The proteomic analysis was independently validated by taking ten identified proteins with relevance to bone aging and confirming their increased abundance in conditioned media from replicatively senescent versus non-senescent MSCs (ACTα2, LTF, SOD1, IL-6, LTBP2, PXDN, SERPINE 1, COL1α1, THBS1, OPG). These target proteins were used to further investigate changes in the MSC SASP profile in response to other inducers of senescence, ionizing radiation (IR) and H2O2. Similar secreted protein expression profiles with replicatively senescent cells were seen with H2O2 treatment except for LTF and PXDN, which were increased by IR treatment. With both IR and H2O2 treatment there was a decrease in THBS1. In vivo investigation of these secreted proteins with aging was shown by significant changes in the abundance of OPG, COL1α1, IL-6, ACTα2, SERPINE 1, and THBS1 in the plasma of aged rats. This unbiased, comprehensive analysis of the changes in the MSC secretome with senescence defines the unique protein signature of the SASP in these cells and provides a better understanding of the aging bone microenvironment

A multi-chamber tissue culture device for load-dependent parallel evaluation of tendon explants

Background Injuries in the musculoskeletal system, such as tendon and ligament ruptures, are challenging to manage and often require surgical reconstructions with limited long-term success. Thus, characterizations of these tissues are urgently needed to better understand cellular mechanisms that regulate tissue homeostasis and healing. Explant culturing systems allow for ex vivo analysis of tissues in an environment that mimics the native microenvironment in vivo. Methods Collaborative efforts within our institution facilitated the establishment of a novel explant culturing system. Tissue specimens cultured in single wells, with individual applied loading and/or biological environment, allowed characterization of tissue cultured under a variety of biological loading conditions. Quantitative PCR analysis for selected gene markers was our primary outcome. Results Data were stratified for analysis by either culture environment or loading condition. Our gene expression results show that specimens clustered by culture condition may differ in molecular markers related to ECM production (e.g., Col1a1, Adamts4) and/or organization (e.g., Tnc, Dnc). In contrast, loading condition did significantly alter the median gene expression levels of tissues in comparison to unloaded control samples, although gene expression values related to ECM degradation (e.g., Mmp1, Mmp10) were altered in tendons cultured under tension in the device. Conclusion Our study demonstrates promising utility of a novel explant culturing system for further characterization of musculoskeletal tissues such as native tendons and ligaments, as well as pathologic fibrotic tissues resulting from arthrofibrosis or Dupuytren’s disease

Melatonin-micronutrients Osteopenia Treatment Study (MOTS): A translational study assessing melatonin, strontium (citrate), vitamin D3 and vitamin K2 (MK7) on bone density, bone marker turnover and health related quality of life in postmenopausal osteopenic women following a one-year double-blind RCT and on osteoblast-osteoclast co-cultures

This one-year double blind randomized control trial assessed the effects of nightly melatonin, strontium (citrate), vitamin D3 and vitamin K2 (MK7; MSDK) on bone mineral density (BMD) and quality of life (QOL) in postmenopausal osteopenic women (ages 49-75). Compared to placebo, MSDK treatment increased BMD in lumbar spine (4.3%) and left femoral neck (2.2%), with an upward trend for total left hip (p=0.069). MSDK increased serum P1NP levels and reduced bone turnover (CTx:P1NP). Psychometric analyses indicated that mood and sleep quality improved for the MSDK group. MSDK-exposed human mesenchymal stem cells (hMSCs) and human peripheral blood monocytes (hPBMCs) plated in transwells or layered demonstrated increases in osteoblastogenesis, decreases in osteoclastogenesis, increases in OPG (TNFRSF11B) and decreases in RANKL (TNFSF11) levels. In transwell osteoblasts, MSDK increased pERK1/2 (MAPK1/MAPK3) and RUNX2 levels; decreased ERK5 (MAPK7); and did not affect the expression of NFκB (NFKB1) and β1integrin (ITGB1). In layered osteoblasts, MSDK also decreased expression of the metabolic proteins PPARγ (PPARG) and GLUT4 (SLC2A4). In adipose-derived human MSCs, MSDK induced osteoblastogenesis. These findings provide both clinical and mechanistic support for the use of MSDK for the prevention or treatment of osteopenia, osteoporosis or other bone-related diseases

Modulating Mesenchymal Stem Cell Behavior Using Human Hair Keratin-Coated Surfaces

Human mesenchymal stem cells (hMSCs) have shown great potential for therapeutic purposes. However, the low frequencies of hMSCs in the body and difficulties in expanding their numbers in vitro have limited their clinical use. In order to develop an alternative strategy for the expansion of hMSCs in vitro, we coated tissue culture polystyrene with keratins extracted from human hair and studied the behavior of cells from 2 donors on these surfaces. The coating resulted in a homogeneous distribution of nanosized keratin globules possessing significant hydrophilicity. Results from cell attachment assays demonstrated that keratin-coated surfaces were able to moderate donor-to-donor variability when compared with noncoated tissue culture polystyrene. STRO-1 expression was either sustained or enhanced on hMSCs cultured on keratin-coated surfaces. This translated into significant increases in the colony-forming efficiencies of both hMSC populations, when the cells were serially passaged. Human hair keratins are abundant and might constitute a feasible replacement for other biomaterials that are of animal origin. In addition, our results suggest that hair keratins may be effective in moderating the microenvironment sufficiently to enrich hMSCs with high colony-forming efficiency ex vivo, for clinical applications

Inhibition of the epigenetic suppressor EZH2 primes osteogenic differentiation mediated by BMP2

Bone-stimulatory therapeutics include bone morphogenetic proteins (e.g. BMP2), parathyroid hormone, and antibody-based suppression of WNT antagonists. Inhibition of the epigenetic enzyme enhancer of zeste homolog 2 (EZH2) is both bone anabolic and osteoprotective. EZH2 inhibition stimulates key components of bone-stimulatory signaling pathways, including the BMP2 signaling cascade. Because of high costs and adverse effects associated with BMP2 use, here we investigated whether BMP2 dosing can be reduced by co-treatment with EZH2 inhibitors. Co-administration of BMP2 with the EZH2 inhibitor GSK126 enhanced differentiation of murine (MC3T3) osteoblasts, reflected by increased alkaline phosphatase activity, Alizarin Red staining, and expression of bone-related marker genes (e.g. Bglap and Phospho1). Strikingly, co-treatment with BMP2 (10 ng/ml) and GSK126 (5 μm) was synergistic and was as effective as 50 ng/ml BMP2 at inducing MC3T3 osteoblastogenesis. Similarly, the BMP2-GSK126 co-treatment stimulated osteogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells, reflected by induction of key osteogenic markers (e.g. Osterix/SP7 and IBSP). A combination of BMP2 (300 ng local) and GSK126 (5 μg local and 5 days of 50 mg/kg systemic) yielded more consistent bone healing than single treatments with either compound in a mouse calvarial critical-sized defect model according to results from μCT, histomorphometry, and surgical grading of qualitative X-rays. We conclude that EZH2 inhibition facilitates BMP2-mediated induction of osteogenic differentiation of progenitor cells and maturation of committed osteoblasts. We propose that epigenetic priming, coupled with bone anabolic agents, enhances osteogenesis and could be leveraged in therapeutic strategies to improve bone mass

Enhancer of zeste homolog 2 (Ezh2) controls bone formation and cell cycle progression during osteogenesis in mice

Epigenetic mechanisms control skeletal development and osteoblast differentiation. Pharmacological inhibition of the histone 3 Lys-27 (H3K27) methyltransferase enhancer of zeste homolog 2 (EZH2) in WT mice enhances osteogenesis and stimulates bone formation. However, conditional genetic loss of Ezh2 early in the mesenchymal lineage (i.e. through excision via Prrx1 promoter– driven Cre) causes skeletal abnormalities due to patterning defects. Here, we addressed the key question of whether Ezh2 controls osteoblastogenesis at later developmental stages beyond patterning. We show that Ezh2 loss in committed pre-osteoblasts by Cre expression via the osterix/Sp7 promoter yields phenotypically normal mice. These Ezh2 conditional knock-out mice (Ezh2 cKO) have normal skull bones, clavicles, and long bones but exhibit increased bone marrow adiposity and reduced male body weight. Remarkably, in vivo Ezh2 loss results in a low trabecular bone phenotype in young mice as measured by micro-computed tomography and histomorphometry. Thus, Ezh2 affects bone formation stage-dependently. We further show that Ezh2 loss in bone marrow– derived mesenchymal cells suppresses osteogenic differentiation and impedes cell cycle progression as reflected by decreased metabolic activity, reduced cell numbers, and changes in cell cycle distribution and in expression of cell cycle markers. RNA-Seq analysis of Ezh2 cKO calvaria revealed that the cyclin-dependent kinase inhibitor Cdkn2a is the most prominent cell cycle target of Ezh2. Hence, genetic loss of Ezh2 in mouse pre-osteoblasts inhibits osteogenesis in part by inducing cell cycle changes. Our results suggest that Ezh2 serves a bifunctional role during bone formation by suppressing osteogenic lineage commitment while simultaneously facilitating proliferative expansion of osteoprogenitor cells