Standardisation of basal medium for reproducible culture of human annulus fibrosus and nucleus pulposus cells.

BACKGROUND: The lifetime prevalence of degenerative disc disease is dramatically high. Numerous investigations on disc degeneration have been performed on cells from annulus fibrosus (AF) and nucleus pulposus (NP) of the intervertebral disc (IVD) in cell culture experiments utilising a broad variety of basal culture media. Although the basal media differ in nutrient formulation, it is not known whether the choice of the basal media itself has an impact on the cell's behaviour in vitro. In this study, we evaluated the most common media used for monolayer expansion of AF and NP cells to set standards for disc cell culture. METHODS: Human AF and NP cells were isolated from cervical discs. Cells were expanded in monolayer until passage P2 using six different common culture media containing alpha-Minimal Essential Medium (alpha-MEM), Dulbecco's Modified Eagle's Medium (DMEM) or Ham's F-12 medium (Ham's F-12) as single medium or in a mixture of two media (alpha/F-12, DMEM/alpha, DMEM/F-12). Cell morphology, cell growth, glycosaminoglycan production and quantitative gene expression of cartilage- and IVD-related markers aggrecan, collagen type II, forkhead box F1 and keratin 18 were analysed. Statistical analysis was performed with two-way ANOVA testing and Bonferroni compensation. RESULTS: AF and NP cells were expandable in all tested media. Both cell types showed similar cell morphology and characteristics of dedifferentiation known for cultured disc cells independently from the media. However, proceeding culture in Ham's F-12 impeded cell growth of both AF and NP cells. Furthermore, the keratin 18 gene expression profile of NP cells was changed in alpha-MEM and Ham's F-12. CONCLUSION: The impact of the different media itself on disc cell's behaviour in vitro was low. However, AF and NP cells were only robust, when DMEM was used as single medium or in a mixture (DMEM/alpha, DMEM/F-12). Therefore, we recommend using these media as standard medium for disc cell culture. Our findings are valuable for the harmonisation of preclinical study results and thereby push the development of cell therapies for clinical treatment of disc degeneration.

Quality Assessment of Surgical Disc Samples Discriminates Human Annulus Fibrosus and Nucleus Pulposus on Tissue and Molecular Level.

A discrimination of the highly specialised annulus fibrosus (AF) and nucleus pulposus (NP) cells in the mature human intervertebral disc (IVD) is thus far still not possible in a reliable way. The aim of this study was to identify molecular markers that distinguish AF and NP cells in human disc tissue using microarray analysis as a screening tool. AF and NP samples were obtained from 28 cervical discs. First, all samples underwent quality sorting using two novel scoring systems for small-sized disc tissue samples including macroscopic, haptic and histological evaluation. Subsequently, samples with clear disc characteristics of either AF or NP that were free from impurities of foreign tissue (IVD score) and with low signs of disc degeneration on cellular level (DD score) were selected for GeneChip analysis (HGU1332P). The 11 AF and 9 NP samples showed distinctly different genome-wide transcriptomes. The majority of differentially expressed genes (DEGs) could be specifically assigned to the AF, whereas no DEG was exclusively expressed in the NP. Nevertheless, we identified 11 novel marker genes that clearly distinguished AF and NP, as confirmed by quantitative gene expression analysis. The novel established scoring systems and molecular markers showed the identity of AF and NP in disc starting material and are thus of great importance in the quality assurance of cell-based therapeutics in regenerative treatment of disc degeneration.

Preclinical good laboratory practice-compliant safety study to evaluate biodistribution and tumorigenicity of a cartilage advanced therapy medicinal product (ATMP).

BACKGROUND: The clinical development of advanced therapy medicinal products (ATMPs), a new class of drugs, requires initial safety studies that deviate from standard non-clinical safety protocols. The study provides a strategy to address the safety aspects of biodistribution and tumorigenicity of ATMPs under good laboratory practice (GLP) conditions avoiding cell product manipulation. Moreover, the strategy was applied on a human ATMP for cartilage repair. METHODS: The testing strategy addresses biodistribution and tumorigenicity using a multi-step analysis without any cell manipulation to exclude changes of test item characteristics. As a safeguard measurement for meeting regulatory expectations, the project design and goals were discussed continuously with the regulatory authority using a staggered scientific advice concept. Subsequently, the strategy was applied to co.don chondrosphere® (huChon spheroid), a tissue-engineered matrix-free ATMP of human normal chondrocytes. In both the biodistribution and tumorigenicity studies, huChon spheroids were implanted subcutaneously into 40 immunodeficient mice. Biodistribution was studied 1 month after implantation. A skin disc containing the huChon spheroid, two surrounding skin rings and selected organs were analyzed by validated, gender-specific, highly-sensitive triplex qPCR and by immunohistochemistry (IHC). RESULTS: No human DNA was detected in distant skin rings and analyzed organs. IHC revealed no direct or indirect indications of cell migration. Tumorigenicity was assessed 6 months after huChon spheroid implantation by palpation, macroscopic inspection, histology and IHC. No mice from the huChon spheroid group developed a tumor at the implantation site. In two mice, benign tumors were detected that were negative for HLA-ABC, suggesting that they were of spontaneous murine origin. CONCLUSIONS: In summary, the presented strategy using a multi-step analysis was confirmed to be suitable for safety studies of ATMPs.

Deregulation of the endogenous C/EBPß LIP isoform predisposes to tumorigenesis.

UNLABELLED: Two long and one truncated isoforms (termed LAP*, LAP, and LIP, respectively) of the transcription factor CCAAT enhancer binding protein beta (C/EBPß) are expressed from a single intronless Cebpb gene by alternative translation initiation. Isoform expression is sensitive to mammalian target of rapamycin (mTOR)-mediated activation of the translation initiation machinery and relayed through an upstream open reading frame (uORF) on the C/EBPß mRNA. The truncated C/EBPß LIP, initiated by high mTOR activity, has been implied in neoplasia, but it was never shown whether endogenous C/EBPß LIP may function as an oncogene. In this study, we examined spontaneous tumor formation in C/EBPß knockin mice that constitutively express only the C/EBPß LIP isoform from its own locus. Our data show that deregulated C/EBPß LIP predisposes to oncogenesis in many tissues. Gene expression profiling suggests that C/EBPß LIP supports a pro-tumorigenic microenvironment, resistance to apoptosis, and alteration of cytokine/chemokine expression. The results imply that enhanced translation reinitiation of C/EBPß LIP promotes tumorigenesis. Accordingly, pharmacological restriction of mTOR function might be a therapeutic option in tumorigenesis that involves enhanced expression of the truncated C/EBPß LIP isoform. KEY MESSAGE: Elevated C/EBPß LIP promotes cancer in mice. C/EBPß LIP is upregulated in B-NHL. Deregulated C/EBPß LIP alters apoptosis and cytokine/chemokine networks. Deregulated C/EBPß LIP may support a pro-tumorigenic microenvironment.

Instruction of mesenchymal cell fate by the transcription factor C/EBPß.

The transcription factor CCAAT/enhancer binding protein beta (C/EBPß) plays a role in the differentiation of a large variety of cell types. C/EBPß was initially described as an early inducer of adipocyte differentiation, however, recent data have shown that this is not the only mesenchymal cell lineage where C/EBPß has an instructive function. Mouse models and tissue culture studies have now established a regulatory role of C/EBPß in osteoblast and in chondrocyte differentiation. These three different cell lineages are derived from the same precursor, the mesenchymal stem cell (MSC). This review will focus on the emerging role of C/EBPß and its different protein isoforms in various mesenchymal cell lineages and its function in adipocyte, chondrocyte and osteoblast differentiation. Moreover, the mesenchymal stem cell has attracted the attention of regenerative medicine in recent years, and the possible role of C/EBPß in this respect will be discussed.

Tal1 regulates osteoclast differentiation through suppression of the master regulator of cell fusion DC-STAMP.

The balance between bone-forming osteoblasts and bone-resorbing osteoclasts is crucial to bone homeostasis, an equilibrium that is disturbed in many bone diseases. The transcription factor Tal1 is involved in the establishment of hematopoietic stem cells in the embryo and is a master regulator of hematopoietic gene expression in the adult. Here, we show that Tal1 is expressed in osteoclasts and that loss of Tal1 in osteoclast progenitors leads to altered expression of >1200 genes. We found that DC-STAMP, a key regulator of osteoclast cell fusion, is a direct target gene of Tal1 and show that Tal1 represses DC-STAMP expression by counteracting the activating function of the transcription factors PU.1 and MITF. The identification of Tal1 as a factor involved in cell fusion contributes to the understanding of osteoclast-associated diseases, including osteoporosis.

Rapamycin inhibits osteoclast formation in giant cell tumor of bone through the C/EBPß - MafB axis.

Giant cell tumor (GCT) of bone is a benign type of tumor, but the presence of hyperactive multinucleated giant osteoclasts cause local osteolytic lesions, increasing morbidity in patients. To specifically target hyperactive multinucleated giant osteoclasts in GCTs, one would envisage the usage of osteoclast inhibitors or genetic modulation of osteoclastogenesis. Recently, we have found that the translationally regulated balance between the transcription factor C/EBPß long (LAP) and short (LIP) protein isoforms regulates osteoclast differentiation. Here, we report that GCTs express high levels of the LIP C/EBPß isoform, which in mice cause giant osteoclast formation. In mice, inhibition of mTOR activity by rapamycin decreased osteoclast differentiation by shifting the alternative translation initiation of C/EBPß isoforms towards LAP. Similarly, rapamycin treatment of GCT cell cultures derived from seven different patients strongly reduced formation of giant osteoclasts and bone resorption. This was accompanied by an increase in MafB, previously shown to be the mediator of the effect of rapamycin on osteoclast differentiation in mice. These data suggest that C/EBPß is a determinant of giant osteoclast formation in GCT and that pharmacological adjustment of the C/EBPß isoform ratio could serve as a potential novel therapeutic approach.

Rapamycin and the transcription factor C/EBPbeta as a switch in osteoclast differentiation: implications for lytic bone diseases.

Lytic bone diseases and in particular osteoporosis are common age-related diseases characterized by enhanced bone fragility due to loss of bone density. Increasingly, osteoporosis poses a major global health-care problem due to the growth of the elderly population. Recently, it was found that the gene regulatory transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) is involved in bone metabolism. C/EBPbeta occurs as different protein isoforms of variable amino terminal length, and regulation of the C/EBPbeta isoform ratio balance was found to represent an important factor in osteoclast differentiation and bone homeostasis. Interestingly, adjustment of the C/EBPbeta isoform ratio by the process of translational control is downstream of the mammalian target of rapamycin kinase (mTOR), a sensor of the nutritional status and a target of immunosuppressive and anticancer drugs. The findings imply that modulating the process of translational control of C/EBPbeta isoform expression could represent a novel therapeutic approach in osteolytic bone diseases, including cancer and infection-induced bone loss.

Glucocorticoids inhibit vascular endothelial growth factor expression in growth plate chondrocytes.

Vascular endothelial growth factor (VEGF) plays an essential role in angiogenesis in the growth plate and ultimately in regulating endochondral ossification. Since longitudinal bone growth is often disturbed in children who are treated with glucocorticoids, we investigated the effects of dexamethasone on VEGF expression by epiphyseal chondrocytes. Cells were cultured from tibial growth plates of neonatal piglets. Using Northern blotting and RT-PCR techniques, the chondrocyte-specific markers aggrecan, collagen II and CD-RAP were detected. Also the glucocorticoid receptor (GR) was expressed. VEGF protein secreted from these cells was examined by ELISA and Western immunoblotting. The VEGF(121) and VEGF(165) isoforms were detected in the supernatant. As determined by RT-PCR, all three major mRNA splice variants were produced, including the species encoding VEGF(189). Dexamethasone (100 nM) inhibited both protein and mRNA expression by approximately 45%. Hydrocortisone (cortisol) and prednisolone also inhibited VEGF secretion, but they were less active than dexamethasone. The inhibitory actions of dexamethasone were almost completely blocked by the GR antagonist Org34116, indicating that the GR mediates these actions. Degradation of the VEGF mRNA was not accelerated by dexamethasone. Therefore, a transcriptional mechanism seems likely. Downregulation of this important growth factor could lead to disruption of the normal invasion of blood vessels in the growth plate, which could contribute to disturbed endochondral ossification and growth.