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BACKGROUND:Bone marrow stromal cells (BMSCs) have classically been cultured in media supplemented with fetal bovine serum (FBS). As an alternative to FBS, pooled solvent detergent apheresis platelets, HPGF-C18, was evaluated for BMSC culture. METHODS:A comparison of passage 2 BMSC growth revealed that 10% HPGF-C18 produced similar cell numbers as 20% FBS. Marrow aspirates from 5 healthy subjects were cultured for 4 passages in 10% HPGF-C18 or 20% FBS and were analyzed for proliferation, colony formation efficiency (CFE), surface marker expression, suppression of mixed lymphocyte reactions (MLRs), global gene and microRNA expression analysis. BMSC supernatant cytokine and growth factor concentrations were also compared. RESULTS:Primary cultures of marrow aspirates in 10% HPGF-C18 and 20% FBS yielded similar numbers and CFE. After 4 passages, 10% HPGF-C18 and 20% FBS yielded similar numbers of BMSCs, surface marker expression patterns and immunosuppression effects. Gene and microRNA expression analysis revealed that BMSCs cultured under the two conditions had distinct expression profiles. Gene Set Enrichment Analysis (GSEA) revealed HPGF-C18-cultured BMSCs were enriched in metabolic processing and biosynthetic pathways, cell proliferation and cell cycle pathways, and immune response pathways. FBS-cultured BMSCs were enriched in MAPK signaling, TGF-beta signaling, cell adhesion and extracellular matrix pathways. Differently expressed microRNAs were related to the osteogenesis of BMSCs. The supernatant of HPGF-C18 BMSCs had higher levels of PEDF and TGFB1 and lower levels of IL6, VEGF, SDF1 and PLGF. CONCLUSIONS:Traditional measures, expansion, surface marker expression and inhibition of MLRs suggest that BMSC cultured in HPGF-C18 and FBS were similar, but analysis at the molecular level revealed many differences. BMSCs cultured in HPGF-C18 should be assessed in specific functional assays that reflect application-specific potency before substituting FBS with HPGF-C18.

For bone marrow stromal cells (BMSC) to be useful in cartilage repair their propensity for hypertrophic differentiation must be overcome. A single day of TGF-β1 stimulation activates intrinsic signaling cascades in BMSCs which subsequently drives both chondrogenic and hypertrophic differentiation. TGF-β1 stimulation upregulates SP7, a transcription factor known to contribute to hypertrophic differentiation, and SP7 remains upregulated even if TGF-β1 is subsequently withdrawn from the chondrogenic induction medium. Herein, we stably transduced BMSCs to express an shRNA designed to silence SP7, and assess the capacity of SP7 silencing to mitigate hypertrophy. SP7 silencing dampened both hypertrophic and chondrogenic differentiation processes, resulting in diminished microtissue size, impaired glycosaminoglycan production and reduced chondrogenic and hypertrophic gene expression. Thus, while hypertrophic features were dampened by SP7 silencing, chondrogenic differentation was also compromised. We further investigated the role of SP7 in monolayer osteogenic and adipogenic cultures, finding that SP7 silencing dampened characteristic mineralization and lipid vacuole formation, respectively. Overall, SP7 silencing affects the trilineage differentiation of BMSCs, but is insufficient to decouple BMSC hypertrophy from chondrogenesis. These data highlight the challenge of promoting BMSC chondrogenesis whilst simultaneously reducing hypertrophy in cartilage tissue engineering strategies. [ABSTRACT FROM AUTHOR]

The tumor suppressor, p53, plays a critical role in suppressing osteosarcoma. Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) have been suggested to give rise to osteosarcomas. However, the role of p53 in BMSCs has not been extensively explored. Here, we report that p53 regulates the lineage choice of mouse BMSCs (mBMSCs). Compared to mBMSCs with wild-type p53, mBMSCs deficient in p53 have enhanced osteogenic differentiation, but with similar adipogenic and chondrogenic differentiation. The role of p53 in inhibiting osteogenic lineage differentiation is mainly through the action of Runx2, a master transcription factor required for the osteogenic differentiation of mBMSCs. We find that p53 indirectly represses the expression of Runx2 by activating the microRNA-34 family, which suppresses the translation of Runx2. Since osteosarcoma may derive from BMSCs, we examined whether p53 has a role in the osteogenic differentiation of osteosarcoma cells and found that osteosarcoma cells with p53 deletion have higher levels of Runx2 and faster osteogenic differentiation than those with wild-type p53. A systems biology approach reveals that p53-deficient mBMSCs are more closely related to human osteosarcoma while mBMSCs with wild-type p53 are similar to normal human BMSCs. In summary, our results indicate that p53 activity can influence cell fate specification of mBMSCs, and provide molecular and cellular insights into the observation that p53 loss is associated with increased osteosarcoma incidence. S tem C ells 2015;33:1304-1319 [ABSTRACT FROM AUTHOR]

In current orthopaedic practice, there is a need to increase the ability to reconstruct large segments of bone lost due to trauma, resection of tumors and skeletal deformities, or when normal regenerative processes have failed such as in non-unions and avascular necrosis. Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells), when used in conjunction with appropriate carriers, represent a means by which to achieve bone regeneration in such cases. While much has been done at the bench and in pre-clinical studies, moving towards clinical application requires the generation of clinical grade cells. What is described herein is an FDA-approved cell manufacturing procedure for the ex vivo expansion of high quality, biologically active human BMSCs. This article is part of a Special Issue entitled Stem Cells and Bone. [ABSTRACT FROM AUTHOR]

Background aims. Bone marrow stromal cells (BMSC) are being used for immune modulatory, anti-inflammatory and tissue engineering applications, but the properties responsible for these effects are not completely understood. Human BMSC were characterized to identify factors that might be responsible for their clinical effects and biomarkers for assessing their quality. Methods. Early passage BMSC prepared from marrow aspirates of seven healthy subjects were compared with three human embryonic stem cell (hESC) samples, CD34++ cells from three healthy subjects and three fibroblast cell lines. The cells were analyzed with oligonucleotide expression microarrays with more than 35 000 probes. Results. BMSC gene expression signatures of BMSC differed from those of hematopoietic stem cells (HSC), hESC and fibroblasts. Genes upregulated in BMSC were involved with cell movement, cell-to-cell signaling and interaction and proliferation. The upregulated genes most probably belonged to pathways for integrin signaling, integrin-linked kinase (ILK) signaling, NF-E2-related factor-2 (NFR2)-mediated oxidative stress response, regulation of actin-based motility by Rho, actin cytoskeletal signaling, caveolar-mediated endocytosis, clathrin-mediated endocytosis and Wingless-type MMTV integration site (Wnt/ββ catenin signaling. Among the most highly upregulated genes were structural extracellular matrix (ECM) proteins (αα5 and ββ5 integrin chains, fibronectin and collagen type IIIαα1 and Vαα1) and functional EMC proteins [connective tissue growth factor (CTGF), transforming growth factor beta-induced protein (TGFBI) and A disintegrin and metalloproteinase (ADAM12)]. Conclusions. Global analysis of human BMSC suggests that they are mobile, metabolically active, proliferative and interactive cells that make use of integrins and integrin signaling. They produce abundant ECM proteins that may contribute to their clinical immune modulatory and anti-inflammatory effects. [ABSTRACT FROM AUTHOR]

Bone formation starts near the end of the embryonic stage of development and continues throughout life during bone modeling and growth, remodeling, and when needed, regeneration. Bone-forming cells, traditionally termed osteoblasts, produce, assemble, and control the mineralization of the type I collagen-enriched bone matrix while participating in the regulation of other cell processes, such as osteoclastogenesis, and metabolic activities, such as phosphate homeostasis. Osteoblasts are generated by different cohorts of skeletal stem cells that arise from different embryonic specifications, which operate in the pre-natal and/or adult skeleton under the control of multiple regulators. In this review, we briefly define the cellular identity and function of osteoblasts and discuss the main populations of osteoprogenitor cells identified to date. We also provide examples of long-known and recently recognized regulatory pathways and mechanisms involved in the specification of the osteogenic lineage, as assessed by studies on mice models and human genetic skeletal diseases. [ABSTRACT FROM AUTHOR]

Background aims. Ex vivo expansion and serial passage of human bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) is required to obtain sufficient quantities for clinical therapy. The BMSC confluence criteria used to determine passage and harvest timing vary widely, and the impact of confluence on BMSC properties remains controversial. The effects of confluence on BMSC properties were studied and confluence-associated markers were identified. Methods. BMSC characteristics were analyzed as they grew from 50% to 100% confluence, including viability, population doubling time, apoptosis, colony formation, immunosuppression, surface marker expression, global gene expression and microRNA expression. In addition, culture supernatant protein, glucose, lactate and pH levels were analyzed. Results. Confluence-dependent changes were detected in the expression of several cell surface markers: 39 culture supernatant proteins, 26 microRNAs and 2078 genes. Many of these surface markers, proteins, microRNAs and genes have been reported to be important in BMSC function. The pigment epithelium-derived factor/vascular endothelial growth factor ratio increased with confluence, but 80% and 100% confluent BMSCs demonstrated a similar level of immunosuppression of mixed lymphocyte reactions. In addition, changes in lactate and glucose levels correlated with BMSC density. Conclusions. BMSC characteristics change as confluence increases. 100% confluent BMSCs may have compromised pro-angiogenesis properties but may retain their immunomodulatory properties. Supernatant lactate and glucose levels can be used to estimate confluence and ensure consistency in passage and harvest timing. Flow cytometry or microRNA expression can be used to confirm that the BMSCs have been harvested at the appropriate confluence. [ABSTRACT FROM AUTHOR]

Abstract: Autologous grafts from axial and appendicular bones commonly used to repair orofacial bone defects often result in unfavorable outcome. This clinical observation, along with the fact that many bone abnormalities are limited to craniofacial bones, suggests that there are significant differences in bone metabolism in orofacial, axial and appendicular bones. It is plausible that these differences are dictated by site-specificity of embryological progenitor cells and osteogenic properties of resident multipotent human bone marrow stromal cells (hBMSCs). This study investigated skeletal site-specific phenotypic and functional differences between orofacial (maxilla and mandible) and axial (iliac crest) hBMSCs in vitro and in vivo. Primary cultures of maxilla, mandible and iliac crest hBMSCs were established with and without osteogenic inducers. Site-specific characterization included colony forming efficiency, cell proliferation, life span before senescence, relative presence of surface markers, adipogenesis, osteogenesis and transplantation in immunocompromised mice to compare bone regenerative capacity. Compared with iliac crest cells, orofacial hBMSCs (OF-MSCs) proliferated more rapidly with delayed senescence, expressed higher levels of alkaline phosphatase and demonstrated more calcium accumulation in vitro. Cells isolated from the three skeletal sites were variably positive for STRO 1, a marker of hBMSCs. OF-MSCs formed more bone in vivo, while iliac crest hBMSCs formed more compacted bone that included hematopoietic tissue and were more responsive in vitro and in vivo to osteogenic and adipogenic inductions. These data demonstrate that hBMSCs from the same individuals differ in vitro and in vivo in a skeletal site-specific fashion and identified orofacial marrow stromal cells as unique cell populations. Further understanding of site-specific properties of hBMSCs and their impact on site-specific bone diseases and regeneration are needed. [Copyright &y& Elsevier]