Improved enrichment of circulating tumor cells from diagnostic leukapheresis product.

The median number of circulating tumor cells (CTCs) detected in 7.5 mL of peripheral blood by CellSearch (PB-CS) in patients with metastatic prostate cancer is in the order of 1-10, which means many samples have insufficient tumor cells for comprehensive characterization. A significant increase is obtained through diagnostic leukapheresis (DLA), however, only 2%-3% of the DLA product can be processed per CellSearch test, limiting the gain. We processed aliquots from 30 DLA products of metastatic prostate cancer patients consisting of 0.2 × 109 leukocytes using CellSearch (DLA-CS) as well as the newly introduced reduced enrichment reagent protocol (RER), which uses 10-fold less enrichment reagents than DLA-CS. The number of tumor cells and the total number of captured cells were determined using the CellTracks Analyzer. Additionally, for six DLA samples, a 1.0 × 109 leukocyte aliquot was processed (RER+), using twofold less enrichment reagents than DLA-CS. A median 2.7-fold reduction in leukocyte co-enrichment was found between DLA-CS and RER methods without any loss in tumor cell recovery (Wilcoxon Signed Ranks Test, p = 0.953). Using 1.0 × 109 leukocyte aliquots a fourfold increase in tumor cells was found compared to DLA-CS and a 19-fold increase compared to PB-CS was obtained. The here-introduced RER protocol results in a higher final sample purity without any loss in tumor cell recovery while using 10-fold less CellSearch capture reagent. With this improved method, 26% of the leukapheresis sample can now be processed using reagents from a single CellSearch test, enabling the obtainment of a sufficient number of CTCs for comprehensive characterization in most metastatic prostate cancer patients.

Leukapheresis increases circulating tumour cell yield in non-small cell lung cancer, counts related to tumour response and survival.

BACKGROUND: Circulating tumour cells (CTCs) can be used to monitor cancer longitudinally, but their use in non-small cell lung cancer (NSCLC) is limited due to low numbers in the peripheral blood. Through diagnostic leukapheresis (DLA) CTCs can be obtained from larger blood volumes. METHODS: Patients with all stages of NSCLC were selected. One total body blood volume was screened by DLA before and after treatment. Peripheral blood was drawn pre- and post DLA for CTC enumeration by CellSearch. CTCs were detected in the DLA product (volume equalling 2 × 108 leucocytes) and after leucocyte depletion (RosetteSep, 9 mL DLA product). Single-cell, whole-genome sequencing was performed on isolated CTCs. RESULTS: Fifty-six patients were included. Before treatment, CTCs were more often detected in DLA (32/55, 58%) than in the peripheral blood (pre-DLA: 18/55, 33%; post DLA: 13/55, 23%, both at p < 0.01). CTCs per 7.5 mL DLA product were median 9.2 times (interquartile range = 5.6-24.0) higher than CTCs in 7.5 mL blood. RosetteSEP did not significantly improve CTC detection (pretreatment: 34/55, 62%, post treatment: 16/34, 47%) and CTCs per mL even decreased compared to DLA (p = 0.04).. Patients with advanced-stage disease with DLA-CTC after treatment showed fewer tumour responses and shorter progression-free survival (PFS) than those without DLA-CTC (median PFS, 2.0 vs 12.0 months, p < 0.01). DLA-CTC persistence after treatment was independent of clinical factors associated with shorter PFS (hazard ratio (HR) = 5.8, 95% confidence interval (CI), 1.4-35.5, p = 0.02). All evaluable CTCs showed aneuploidy. CONCLUSIONS: DLA detected nine times more CTCs than in the peripheral blood. The sustained presence of CTCs in DLA after treatment was associated with therapy failure and shortened PFS. TRIAL REGISTRATION: The study was approved by the Medical Ethical Committee (NL55754.042.15) and was registered in the Dutch trial register (NL5423).

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

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.

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.

Measuring antigen expression of cancer cell lines and circulating tumour cells.

When evaluating EpCAM-based enrichment technologies for circulating tumour cells (CTCs), the cell lines used should closely resemble real CTCs, meaning the EpCAM expression of CTCs needs to be known, but also the EpCAM expression of cell lines at different institutions and times is important. As the number of CTCs in the blood is low, we enriched CTCs through the depletion of leukocytes from diagnostic leukapheresis products of 13 prostate cancer patients and measured EpCAM expression using quantitative flow cytometry. Antigen expression was compared between multiple institutions by measuring cultures from each institution. Capture efficiency was also measured for one of the used cell lines. Results show CTCs derived from castration-sensitive prostate cancer patients have varying but relatively low EpCAM expression, with median expression per patient ranging from 35 to 89,534 (mean 24,993) molecules per cell. A large variation in the antigen expression of identical cell lines cultured at different institutions was found, resulting in recoveries when using the CellSearch system ranging from 12 up to 83% for the same cell line. We conclude that large differences in capture efficiency can occur while using the same cell line. To closely resemble real CTCs from castration-sensitive prostate cancer patients, a cell line with a relatively low EpCAM expression should be used, and its expression should be monitored frequently.

Flow-based immunomagnetic enrichment of circulating tumor cells from diagnostic leukapheresis product.

The clinical utility of circulating tumor cells (CTCs) is hampered by the low number of cells detected. Diagnostic leukapheresis (DLA) offers a solution but, due to the observed non-specific binding and clumping, processing of DLA samples using the CellSearch system only allows for the processing of aliquots consisting of ~ 2% of the total DLA sample per test. Here, we introduce a flow enrichment target capture Halbach-array (FETCH)-based separation method in combination with a DNase preprocessing step to capture CTCs from larger fractions of DLA products without clumping. To evaluate the FETCH method, we processed peripheral blood samples from 19 metastatic castration-naïve prostate cancer (mCNPC) patients with CellSearch, and processed 2% aliquots of leukapheresis samples from the same patients with CellSearch as well as FETCH with or without DNase preprocessing. Using 2% aliquots from six patients, the use of FETCH with fewer immunomagnetic epithelial cellular adhesion molecule (EpCAM) conjugated ferrofluids was tested, whereas 20% aliquots from four patients were used to evaluate the processing of 10-fold larger DLA samples using FETCH. Results show that the cell clumping normally seen after immunomagnetic enrichment of DLA material was greatly reduced with the use of DNase pretreatment, while the number of CTCs detected was not affected. The number of CTCs detected in 2% aliquots of DLA using FETCH was unchanged compared to CellSearch and did not decrease when using down to 10% of the volume of immunomagnetic anti-EpCAM ferrofluids normally used in a CellSearch test, whereas the number of co-enriched white blood cells reduced a median 3.2-fold. Processing of a 20% aliquot of DLA with FETCH resulted in a 14-fold increase in CTCs compared to the processing of 2% aliquots of DLA using CellSearch and a total 42-fold median increase in CTCs compared to peripheral-blood CellSearch.

Optimal Halbach Configuration for Flow-through Immunomagnetic CTC Enrichment.

Due to the low frequency of circulating tumor cells (CTC), the standard CellSearch method of enumeration and isolation using a single tube of blood is insufficient to measure treatment effects consistently, or to steer personalized therapy. Using diagnostic leukapheresis this sample size can be increased; however, this also calls for a suitable new method to process larger sample inputs. In order to achieve this, we have optimized the immunomagnetic enrichment process using a flow-through magnetophoretic system. An overview of the major forces involved in magnetophoretic separation is provided and the model used for optimizing the magnetic configuration in flow through immunomagnetic enrichment is presented. The optimal Halbach array element size was calculated and both optimal and non-optimal arrays were built and tested using anti-EpCAM ferrofluid in combination with cell lines of varying EpCAM antigen expression. Experimentally measured distributions of the magnetic moment of the cell lines used for comparison were combined with predicted recoveries and fit to the experimental data. Resulting predictions agree with measured data within measurement uncertainty. The presented method can be used not only to optimize magnetophoretic separation using a variety of flow configurations but could also be adapted to optimize other (static) magnetic separation techniques.

High throughput surface plasmon resonance imaging method for clinical detection of presence and strength of binding of IgM, IgG and IgA antibodies against SARS-CoV-2 during CoViD-19 infection.

Surface Plasmon Resonance imaging (SPRi) was used to determine the presence and strength of binding of IgG, IgM and IgA against the Receptor Binding Domain (RBD) of SARS-CoV-2 in sera of 102 CoViD-19 and non-CoViD-19 patients. The SPRi assay simultaneously measures the antibody isotype levels and the strength of binding to the RBD of ultimate 384 patient samples in one run. It turns out that during the course of the disease, the IgG levels and strength of binding increased while generally the IgM and IgA levels go down. Recovered patients all show high strength of binding of the IgG type to the RBD protein. The anti-RBD immunoglobulins SPRi assay provides additional insights in the immune status of patients recovering from CoViD-19. This new high throughput method can be applied for the assessment of the quality of the immune reaction of healthy individuals to SARS-CoV-2 and its mutants in vaccination programs.•Surface Plasmon Resonance imaging is an unprecedented technology for high throughput screening of antibody profiling of CoViD19 patients.•Fingerprinting of isotypes IgM, IgG and IgA can be performed for 384 patients in one run.•An affinity maturation effect was shown for patients recovering from CoViD19.

Presence and strength of binding of IgM, IgG and IgA antibodies against SARS-CoV-2 during CoViD-19 infection.

Surface Plasmon Resonance imaging (SPRi) was used to determine the presence and strength of binding of IgG, IgM and IgA against the Receptor Binding Domain (RBD) of SARS-CoV-2 in sera of 119 CoViD-19 patients. The SPRi assay measures the antibody isotype levels and the strength of binding to the RBD of ultimate 384 patient samples in one run. It turns out that during the course of the disease, the IgG levels and strength of binding increased while generally the IgM and IgA levels go down. Recovered patients all show high strength of binding of the IgG type to the RBD protein. The anti-RBD immunoglobulins SPRi assay provides additional insights in the immune status of patients recovering from CoViD-19 and this new method can furthermore be applied for the assessment of the quality of the immune reaction of healthy individuals to SARS-CoV-2 in vaccination programs.

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

BACKGROUND: Oligonucleotide 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. RESULTS: We 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. CONCLUSIONS: When 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.

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.

Detection of circulating tumor cells in blood of pancreatic ductal adenocarcinoma patients.

Aim: Previous studies suggest that circulating tumor cells (CTC) are present at very low frequencies in blood of pancreatic cancer (PC) patients. However, no technique has proven efficient for their detection, in part due to the lack of accurate tumor markers. Here, we evaluated the potential utility of two marker candidates - Mucin 16 (MUC16) and Tetraspanin 1 (TSPAN1) - identified through a detailed review of the literature. Methods: To evaluate the pattern of expression of both markers in pancreatic tumor cells vs. normal blood, we used cell lines derived from pancreatic cancer patients and blood from healthy adults. Results: Antibodies against both MUC16 and TSPAN1 showed expression in three pancreatic cancer (PC) cell lines while they were absent in blood cells. To evaluate the efficiency of isolating tumor cells from blood, PC cell lines were spiked at different frequencies in blood, sequentially stained with biotin-conjugated TSPAN1 and MUC16 antibodies and a streptavidin ferrofluids, followed by immunomagnetic enrichment. The recovery of spiked TSPAN1+ tumor cells was high with limited contamination by leukocytes. In contrast, no PC cells were isolated when the biotin MUC16 reagent was used because the biotin-conjugated clone did not recognize PC cells. Conclusion: The combination of MUC16, TSPAN1, and epithelial cell adhesion molecule (EpCAM) antibodies will likely increase the efficiency of capturing circulating tumor cell in blood of pancreatic ductal adenocarcinoma. To further develop a protocol for isolation of circulating tumor cell in blood of PC patients, high amounts of antibodies (5-10 mg) against EpCAM, MUC16, and TSPAN1 will be needed.

Designed Surface Topographies Control ICAM-1 Expression in Tonsil-Derived Human Stromal Cells.

Fibroblastic reticular cells (FRCs), the T-cell zone stromal cell subtype in the lymph nodes, create a scaffold for adhesion and migration of immune cells, thus allowing them to communicate. Although known to be important for the initiation of immune responses, studies about FRCs and their interactions have been impeded because FRCs are limited in availability and lose their function upon culture expansion. To circumvent these limitations, stromal cell precursors can be mechanotranduced to form mature FRCs. Here, we used a library of designed surface topographies to trigger FRC differentiation from tonsil-derived stromal cells (TSCs). Undifferentiated TSCs were seeded on a TopoChip containing 2176 different topographies in culture medium without differentiation factors, then monitored cell morphology and the levels of ICAM-1, a marker of FRC differentiation. We identified 112 and 72 surfaces that upregulated and downregulated, respectively, ICAM-1 expression. By monitoring cell morphology, and expression of the FRC differentiation marker ICAM-1 via image analysis and machine learning, we discovered correlations between ICAM-1 expression, cell shape and design of surface topographies and confirmed our findings by using flow cytometry. Our findings confirmed that TSCs are mechano-responsive cells and identified particular topographies that can be used to improve FRC differentiation protocols.

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.

Effect of antioxidant supplementation on the total yield, oxidative stress levels, and multipotency of bone marrow-derived human mesenchymal stromal cells.

Bone marrow-derived multipotent mesenchymal stromal cells (MSCs) are the most frequently investigated cell type for potential regenerative strategies because they are relatively easy to isolate and are able to differentiate into several mesenchymal lineages. Unfortunately, during ex vivo culture, MSCs present gradual loss of differentiation potential and reduced clinical efficacy. Reactive oxygen species (ROS) are associated with oxidative damage and accumulate during MSC expansion. Because ROS are believed to be involved in the loss of multipotency, we hypothesized that compounds with antioxidant activity have the capacity to scavenge ROS, prevent cellular damage, and rescue culture-induced loss of multipotency. In this manuscript, we show that antioxidant supplementation can partially rescue the loss of alkaline phosphatase expression induced by oxidizing agents and increases the yield of hMSCs, when supplemented to a fresh bone marrow aspirate. Concomitantly, oxidative DNA damage and ROS levels in hMSCs were reduced by antioxidants. We conclude that antioxidant supplementation during MSC expansion reduces the DNA damage load and increases the MSC yield.

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.

The effect of donor variation and senescence on endothelial differentiation of human mesenchymal stromal cells.

Application of autologous cells is considered for a broad range of regenerative therapies because it is not surrounded by the immunological and ethical issues of allo- or xenogenic cells. However, isolation, expansion, and application of autologous cells do suffer from variability in therapeutic efficacy due to donor to donor differences and due to prolonged culture. One important source of autologous cells is mesenchymal stromal cells (MSCs), which can differentiate toward endothelial-like cells, thus making them an ideal candidate as cell source for tissue vascularization. Here we screened MSCs from 20 donors for their endothelial differentiation capacity and correlated it with the gene expression profile of the whole genome in the undifferentiated state. Cells of all donors were able to form tubes on Matrigel and induced the expression of endothelial genes, although with quantitative differences. In addition, we analyzed the effect of prolonged in vitro expansion on the multipotency of human MSCs and found that endothelial differentiation is only mildly sensitive to expansion-induced loss of differentiation as compared to osteogenic and adipogenic differentiation. Our results show the robustness of the endothelial differentiation protocol and the gene expression data give insight in the differences in endothelial differentiation between donors.

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.

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.