Anouk Mentink

A mesenchymal stromal cell gene signature for donor age

Human aging is associated with loss of function and regenerative capacity. Human bone marrow derived mesenchymal stromal cells (hMSCs) are involved in tissue regeneration, evidenced by their capacity to differentiate into several lineages and therefore are considered the golden standard for cell-based regeneration therapy. Tissue maintenance and regeneration is dependent on stem cells and declines with age and aging is thought to influence therapeutic efficacy, therefore, more insight in the process of aging of hMSCs is of high interest. We, therefore, hypothesized that hMSCs might reflect signs of aging. In order to find markers for donor age, early passage hMSCs were isolated from bone marrow of 61 donors, with ages varying from 17–84, and clinical parameters, in vitro characteristics and microarray analysis were assessed. Although clinical parameters and in vitro performance did not yield reliable markers for aging since large donor variations were present, genome-wide microarray analysis resulted in a considerable list of genes correlating with human age. By comparing the transcriptional profile of aging in human with the one from rat, we discovered follistatin as a common marker for aging in both species. The gene signature presented here could be a useful tool for drug testing to rejuvenate hMSCs or for the selection of more potent, hMSCs for cell-based therapy.

H3K4me1 marks DNA regions hypomethylated during aging in human stem and differentiated cells

In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type-independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors.

Predicting the therapeutic efficacy of MSC in bone tissue engineering using the molecular marker CADM1

Mesenchymal stromal cells (hMSCs) are advancing into the clinic but the therapeutic efficacy of hMSCs faces the problem of donor variability. In bone tissue engineering, no reliable markers have been identified which are able to predict the bone-forming capacity of hMSCs prior to implantation. To this end, we isolated hMSCs from 62 donors and characterized systematically their in vitro lineage differentiation capacity, gene expression signature and in vivo capacity for ectopic bone formation. Our data confirms the large variability of in vitro differentiation capacity which did not correlate with in vivo ectopic bone formation. Using DNA microarray analysis of early passage hMSCs we identified a diagnostic bone-forming classifier. In fact, a single gene, CADM1, strongly correlated with the bone-forming capacity of hMSCs and could be used as a reliable in vitro diagnostic marker. Furthermore, data mining of genes expressed correlating with in vivo bone formation represented involvement in neurogenic processes and Wnt signaling. We will apply our data set to predict therapeutic efficacy of hMSCs and to gain novel insight in the process of bone regeneration. Our bio-informatics driven approach may be used in other fields of cell therapy to establish diagnostic markers for clinical efficacy.

Monolithic and assembled polymer-ceramic composites for bone regeneration

The rationale for the use of polymer-ceramic composites for bone regeneration stems from the natural composition of bone, with collagen type I and biological apatite as the main organic and inorganic constituents, respectively. In the present study composite materials of PolyActive™ (PA), a poly(ethylene oxide terephthalate)/poly(butylene terephtalate) co-polymer, and hydroxyapatite (HA) at a weight ratio of 85:15 were prepared by rapid prototyping (RP) using two routes. In the first approach pre-extruded composite filaments of PA-HA were processed using three-dimensional fibre deposition (3DF) (conventional composite scaffolds). In the second approach PA scaffolds were fabricated using 3DF and combined with HA pillars produced inside stereolithographic moulds that fitted inside the pores of the PA three-dimensional structure (assembled composite scaffolds). Analysis of calcium and phosphate release in a simulated physiological solution, not containing calcium or phosphate, revealed significantly higher values for the HA pillars compared with other scaffolds. Release in simulated body fluid saturated with respect to HA did not show significant differences among the different scaffolds. Human mesenchymal stromal cells were cultured on polymer (3DF), conventional composite (3DF-HA) and assembled composite (HA assembled in 3DF) scaffolds and assessed for morphology, metabolic activity, DNA amount and gene expression of osteogenic markers using real time quantitative polymerase chain reaction (PCR). Scanning electron microscopy images showed that the cells attached to and infiltrated all the scaffolds. Assembled composites had a higher metabolic activity compared with 3DF-HA scaffolds while no significant differences were observed in DNA amounts. Gene expression of osteopontin in the assembled composite was significantly higher compared with the conventional composites. The strategy of composite fabrication by assembly appears to be a promising alternative to the conventional composite fabrication route for scaffolds for bone regeneration.

Capture of Tumor Cells on Anti-EpCAM-Functionalized Poly(acrylic acid)-Coated Surfaces

The presence of tumor cells in blood is predictive of short survival in several cancers and their isolation and characterization can guide toward the use of more effective treatments. These circulating tumor cells (CTC) are, however, extremely rare and require a technology that is sufficiently sensitive and specific to identify CTC against a background of billions of blood cells. Immuno-capture of cells expressing the epithelial cell adhesion molecule (EpCAM) are frequently used to enrich CTC from blood. The choice of bio conjugation strategy and antibody clone is crucial for adequate cell capture but is poorly understood. In this study, we determined the binding affinity constants and epitope binding of the EpCAM antibodies VU1D-9, HO-3, EpAb3-5, and MJ-37 by surface plasmon resonance imaging (SPRi). Glass surfaces were coated using a poly(acrylic acid) based coating and functionalized with anti-EpCAM antibodies. Binding of cells from the breast carcinoma cell line (SKBR-3) to the functionalized surfaces were compared. Although EpAb3-5 displayed the highest binding affinity HO-3 captured the highest amount of cells. Hence we report differences in the performance of the different antibodies and more importantly that the choice of antibody to capture CTC should be based on multiple assays.

The effect of PKC activation and inhibition on osteogenic differentiation of human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are being considered for several areas of clinical therapy, due to their multipotent nature. For instance, osteogenic hMSCs are applied in bone tissue engineering, but current differentiation protocols need further optimization before they can be clinically applied. Protein kinase C (PKC) family members have been implicated in bone metabolism, which prompted us to use a pharmaceutical approach to manipulate PKC signalling in hMSCs. Inhibition of PKC resulted in a dose‐dependent inhibition of dexamethasone‐induced osteogenic differentiation. Surprisingly, PKC activation using phorbol 12‐myristate 13‐acetate (PMA) also resulted in inhibition of osteogenesis, although we observed that inhibition was more pronounced at low than at high concentrations of PMA. Furthermore, we observed that inhibition of PKCδ blocked alkaline phosphatase (ALP, an early marker of osteogenic differentiation) expression, whereas inhibition of the conventional PKC subfamily and PKCµ using Gö6976 resulted in an induction of ALP activity, collagen (I) expression and mineralization. In conclusion, inhibition of the conventional PKCs/PKCµ and activation of PKCδ could further benefit osteogenic differentiation of hMSCs in vitro and in vivo, which is currently under investigation. Copyright

Effect of Chordin-Like 1 on MC3T3-E1 and Human Mesenchymal Stem Cells

Since the discovery that bone morphogenetic proteins (BMPs) are able to induce ectopic bone formation, considerable effort has been devoted to apply it for bone regeneration. BMP activity needs to be temporally and spatially controlled and the organism has devised ways to achieve it. Here we show that the BMP inhibitor chordin-like 1 can interfere with BMP2 signalling thereby affecting the osteogenic differentiation of MC3T3-E1 cells. Besides its function as a BMP antagonist, chordin-like 1 enhanced the proliferation of human mesenchymal stem cells (hMSCs) in a BMP2-independent manner. When MC3T3-E1 cells were exposed to recombinant chordin-like 1 there was an inhibition of alkaline phosphatase (ALP) expression, whereas in the case of hMSCs no effect was observed. However, chordin-like 1 dose-dependently increased the proliferation of hMSCs. This effect is probably BMP2 independent because the chordin-like 1 concentration that stimulates proliferation does not interfere with BMP signalling monitored by a Smad-dependent reporter gene. Our data point towards a novel, BMP-independent role of chordin-like 1 in hMSC proliferation.

Delineation of amplification, hybridization and location effects in microarray data yields better-quality normalization

BackgroundOligonucleotide arrays have become one of the most widely used high-throughput tools in biology. Due to their sensitivity to experimental conditions, normalization is a crucial step when comparing measurements from these arrays. Normalization is, however, far from a solved problem. Frequently, we encounter datasets with significant technical effects that currently available methods are not able to correct.ResultsWe show that by a careful decomposition of probe specific amplification, hybridization and array location effects, a normalization can be performed that allows for a much improved analysis of these data. Identification of the technical sources of variation between arrays has allowed us to build statistical models that are used to estimate how the signal of individual probes is affected, based on their properties. This enables a model-based normalization that is probe-specific, in contrast with the signal intensity distribution normalization performed by many current methods. Next to this, we propose a novel way of handling background correction, enabling the use of background information to weight probes during summarization. Testing of the proposed method shows a much improved detection of differentially expressed genes over earlier proposed methods, even when tested on (experimentally tightly controlled and replicated) spike-in datasets.ConclusionsWhen a limited number of arrays are available, or when arrays are run in different batches, technical effects have a large influence on the measured expression of genes. We show that a detailed modelling and correction of these technical effects allows for an improved analysis in these situations.

Toward a real liquid biopsy in metastatic breast and prostate cancer: Diagnostic LeukApheresis increases CTC yields in a European prospective multicenter study (CTCTrap): Toward a real liquid biopsy in metastatic breast and prostate cancer

Frequently, the number of circulating tumor cells (CTC) isolated in 7.5 mL of blood is too small to reliably determine tumor heterogeneity and to be representative as a “liquid biopsy”. In the EU FP7 program CTCTrap, we aimed to validate and optimize the recently introduced Diagnostic LeukApheresis (DLA) to screen liters of blood. Here we present the results obtained from 34 metastatic cancer patients subjected to DLA in the participating institutions. About 7.5 mL blood processed with CellSearch® was used as “gold standard” reference. DLAs were obtained from 22 metastatic prostate and 12 metastatic breast cancer patients at four different institutions without any noticeable side effects. DLA samples were prepared and processed with different analysis techniques. Processing DLA using CellSearch resulted in a 0–32 fold increase in CTC yield compared to processing 7.5 mL blood. Filtration of DLA through 5 μm pores microsieves was accompanied by large CTC losses. Leukocyte depletion of 18 mL followed by CellSearch yielded an increase of the number of CTC but a relative decrease in yield (37%) versus CellSearch DLA. In four out of seven patients with 0 CTC detected in 7.5 mL of blood, CTC were detected in DLA (range 1–4 CTC). The CTC obtained through DLA enables molecular characterization of the tumor. CTC enrichment technologies however still need to be improved to isolate all the CTC present in the DLA.

Abstract 1532: The isolation of CTC from diagnostic leukapheresis

Introduction At present, the CellSearch system is the only validated method for the detection of circulating tumor cells (CTC) that has been cleared by the U.S. Food and Drug Administration. This system, designed for the enumeration of CTC in 7.5 mL of blood, detects CTC based on their expression of EpCAM and cytokeratins and negativity for CD45. However, the number of CTC that are detected in patients with metastatic carcinomas is in most cases too small to reliably determine tumor heterogeneity and to be representative as a ‘liquid biopsy’. Our aim is to identify and isolate a sufficient number of circulating tumor cells in virtually all metastatic cancer patients to enable their characterization and to represent a real-time liquid biopsy. For this purpose we used Diagnostic LeukApheresis (DLA) to increase the blood volume to be analyzed. We developed several techniques to isolate CTC from DLA to enable a multicenter comparison of CTC detection in DLA products. Methods DLAs were performed for ∼1 hour to obtain 40 mL of product containing ∼4 x10⁁9 mononuclear cells representing ∼1 liter of blood. Using CellSearch a maximum of 2 mL of DLA could be processed for EpCAM+ CTC (Fisher et al. doi: 10.1073/pnas.1313594110) and EpCAM- CTC (de Wit et al doi: 10.1038/srep12270). Using filtration through microsieves with 5 μm pores a maximum of 1.0 mL of DLA could be processed. To process 18 mL DLA product protocols were developed for leukocyte depletion using RosetteSep™ (StemCell Technologies, USA) and for EpCAM selection using an anti-EpCAM coated column (Leukocare AG). All enriched cell fractions were stained using CD45 PerCP, Cytokeratins PE and the nuclear dye DAPI, followed by fluorescence microscopy scanning and analysis. Results Leukocyte depletion using the RosetteSep™ CTC Enrichment cocktail was first optimized using small sample volumes (1 mL) spiked with cells from cancer cell lines. Depletion of leukocytes ranged from 3.1 to 3.9 logs with an average recovery of spiked cancer cells of 50-60%. Isolation of CTC expressing EpCAM was pursued using anti-EpCAM coated columns and optimized for selection and release of EpCAM expressing cells by passage of cells from cancer cell lines through the column resulting in 34-100% recovery. Both procedures were scaled up to enable processing of 18 mL of DLA. Leukocytes were depleted using RosetteSepTM by 3.1 - 3.9 logs whereas with anti-EpCAM columns only 1.7 - 1.8 logs depletion were reached. Using RosetteSepTM 21% and with the anti-EpCAM coated columns 2% of the tumor cells spiked into 18ml DLA were recovered. Conclusion Standard operating procedures were developed to isolate CTC in DLA9s from breast, prostate cancer and lung cancer patients for evaluation and comparison in the EU sponsored consortiums CTCTrap (www.utwente.nl/tnw/ctctrap/) and CANCER-ID (www.CANCER-ID.eu). Isolation of EpCAM expressing CTC using the anti-EpCAM coated columns will need further optimization before it can proceed to multicenter comparison. Citation Format: Kiki C. Andree, Anouk Mentink, Martin Scholz, Roland Kirchner, Rui P. Neves, Christiane Driemel, Rita Lampignano, Hans Neubauer, Dieter Niederacher, Tanja Fehm, Wolfram T. Knoefel, Johannes C. Fischer, Nikolas H. Stoecklein, Leon WMM Terstappen. The isolation of CTC from diagnostic leukapheresis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1532.