Health technology assessment-based approach to flow cytometric immunophenotyping of acute leukemias: a literature classification.

OBJECTIVE: Acute leukemia (AL) is a broad, heterogeneous group of malignant diseases. The diagnostic workup of AL is based on several clinical and laboratory findings, including flow cytometric immunophenotyping. However, the role of this assay in the diagnosis of AL has not been systematically investigated. The aim of this study was to determine the accuracy and utility of flow cytometric immunophenotyping in the identification, characterization, and staging of AL. METHODS: We performed a systematic selection and classification of the literature since 1980, focused on flow cytometric immunophenotyping of AL. We applied a 6-variables model to cover both the technical capabilities and the clinical value of flow cytometric immunophenotyping in the diagnosis of AL. RESULTS: Using 3 key words (acute leukemia, immunophenotyping, flow cytometry), we screened the literature from January 1985 to April 2015 in PubMed and Embase databases and found 1010 articles. A total of 363 were selected and submitted to the expert panel, which selected a final data set of 248 articles to be analyzed. Of these, 160 were focused on clinical and biological issues, 55 were technical articles, and 31 were reviews. These 248 articles were then analyzed according to the 6-variables model and definitively classified. CONCLUSIONS: We assessed the literature on flow cytometric immunophenotyping of AL over 3 decades as the first step toward an evidence-based analysis of the impact of this technology on the clinical management of patients with AL.

Counteracting roles of MHCI and CD8+ T cells in the peripheral and central nervous system of ALS SOD1G93A mice.

BACKGROUND: The major histocompatibility complex I (MHCI) is a key molecule for the interaction of mononucleated cells with CD8+T lymphocytes. We previously showed that MHCI is upregulated in the spinal cord microglia and motor axons of transgenic SOD1G93A mice. METHODS: To assess the role of MHCI in the disease, we examined transgenic SOD1G93A mice crossbred with ß2 microglobulin-deficient mice, which express little if any MHCI on the cell surface and are defective for CD8+ T cells. RESULTS: The lack of MHCI and CD8+ T cells in the sciatic nerve affects the motor axon stability, anticipating the muscle atrophy and the disease onset. In contrast, MHCI depletion in resident microglia and the lack of CD8+ T cell infiltration in the spinal cord protect the cervical motor neurons delaying the paralysis of forelimbs and prolonging the survival of SOD1G93A mice. CONCLUSIONS: We provided straightforward evidence for a dual role of MHCI in the peripheral nervous system (PNS) compared to the CNS, pointing out regional and temporal differences in the clinical responses of ALS mice. These findings offer a possible explanation for the failure of systemic immunomodulatory treatments and suggest new potential strategies to prevent the progression of ALS.

Trabectedin modulates the senescence-associated secretory phenotype and promotes cell death in senescent tumor cells by targeting NF-κB.

Therapy-induced senescence is a major cellular response to chemotherapy in solid tumors. Senescent tumor cells acquire a secretory phenotype, or SASP, and produce pro-inflammatory factors, whose expression is largely under NF-κB transcriptional control. Secreted factors play a positive role in driving antitumor immunity, but also exert negative influences on the microenvironment, and promote tumor growth and metastasis. Moreover, subsets of cancer cells can escape the senescence arrest, driving tumor recurrence after treatments. Hence, removal the senescent tumor cells, or reprogramming of the senescent secretome, have become attractive therapeutic options. The marine drug trabectedin was shown to inhibit the production of pro-inflammatory mediators by tumor-infiltrating immune cells and by myxoid liposarcoma cells. Here, we demonstrate that trabectedin inhibits the SASP, thus limiting the pro-tumoral activities of senescent tumor cells in vitro. We show that trabectedin modulates NF-κB transcriptional activity in senescent tumor cells. This results in disruption of the balance between antiapoptotic and proapoptotic signals, and sensitization of cells to Fas-mediated apoptosis. Further, we found that trabectedin inhibits escape from therapy-induced senescence, at concentrations that do not affect the viability of bulk tumor population. Overall, our data demonstrate that trabectedin has the potential to inhibit multiple detrimental effects of therapy-induced senescence.

Tumor progression and metastatic dissemination in ovarian cancer after dose-dense or conventional paclitaxel and cisplatin plus bevacizumab.

The efficacy of therapeutic regimens incorporating weekly or every-3-weeks paclitaxel (PTX) for ovarian cancer is debated. We investigated the addition of bevacizumab in regimens of chemotherapy with different PTX doses and schedules in preclinical models. Treatments were cisplatin (DDP) with weekly PTX (conventional), or dose-dense-equi (every other day to the conventional cumulative dose), or dose-dense-high (total dose 1.5 times higher), with or without bevacizumab. Treatment efficacy was evaluated analyzing tumor growth in different time-windows in two patient-derived ovarian cancer xenografts with different sensitivity to cisplatin. Tumor progression, metastasis and survival were studied in ovarian cancer models growing orthotopically and disseminating in the mouse peritoneal cavity. Short-term effects on cell cycle, tumor cell proliferation/apoptosis and vasculature were evaluated by flow cytometry and immunohistochemistry. PTX dose-dense (with/without DDP) was superior to the conventional scheme in a dose-dependent manner; the high efficacy was confirmed by the lower ratio of tumor to normal cells. All schemes benefited from bevacizumab, which reduced tumor vessels. However, DDP/PTX dose-dense-high (only chemotherapy) was at least as active as DDP/PTX conventional plus bevacizumab. DDP/PTX dose-dense-high plus bevacizumab was the most effective in delaying tumor progression, though it did not prolong mouse survival and the continuous treatment with bevacizumab was associated with a malignant disease. These findings indicate that the effect of bevacizumab in combination with chemotherapy may depend on the schedule-dose of the treatment and help to explain the unclear benefits after bevacizumab.

Application of 3D Mass Spectrometry Imaging to TKIs.

Mass spectrometry imaging (MSI) allows visualization of endogenous and exogenous compound in tissue sections based on its molecular mass. The 3D reconstruction by MSI provides a more informative description of the tumor drug distribution compared to the high-performance liquid chromatography method, highlighting the heterogeneity of intratumor drug concentration. This additional information can be important in understanding chemoresistance to target agents. Here, we present the 3D visualization of the tyrosine kinase inhibitor (TKI), imatinib, in a xenograft model of resistant malignant pleural mesothelioma.

FOXM1 expression is significantly associated with chemotherapy resistance and adverse prognosis in non-serous epithelial ovarian cancer patients.

BACKGROUND: Epithelial ovarian cancer (EOC) is a spectrum of different diseases, which makes their treatment a challenge. Forkhead box M1 (FOXM1) is an oncogene aberrantly expressed in many solid cancers including serous EOC, but its role in non-serous EOCs remains undefined. We examined FOXM1 expression and its correlation to prognosis across the three major EOC subtypes, and its role in tumorigenesis and chemo-resistance in vitro. METHODS: Gene signatures were generated by microarray for 14 clear-cell and 26 endometrioid EOCs, and 15 normal endometrium snap-frozen biopsies. Validation of FOXM1 expression was performed by RT-qPCR and immunohistochemistry in the same samples and additionally in 50 high-grade serous EOCs and in their most adequate normal controls (10 luminal fallopian tube and 20 ovarian surface epithelial brushings). Correlations of FOXM1 expression to clinic-pathological parameters and patients' prognosis were evaluated by Kaplan-Meier and Cox proportional-hazards analyses. OVCAR-3 and two novel deeply characterized EOC cell lines (EOC-CC1 and OSPC2, with clear-cell and serous subtype, respectively) were employed for in vitro studies. Effects of FOXM1 inhibition by transient siRNA transfection were evaluated on cell-proliferation, cell-cycle, colony formation, invasion, and response to conventional first- and second-line anticancer agents, and to the PARP-inhibitor olaparib. Gene signatures of FOXM1-silenced cell lines were generated by microarray and confirmed by RT-qPCR. RESULTS: A significant FOXM1 mRNA up-regulation was found in EOCs compared to normal controls. FOXM1 protein overexpression significantly correlated to serous histology (p = 0.001) and advanced FIGO stage (p = 0.004). Multivariate analyses confirmed FOXM1 protein overexpression as an independent indicator of worse disease specific survival in non-serous EOCs, and of shorter time to progression in platinum-resistant cases. FOXM1 downregulation in EOC cell lines inhibited cell growth and clonogenicity, and promoted the cytotoxic effects of platinum compounds, doxorubicin hydrochloride and olaparib. Upon FOXM1 knock-down in EOC-CC1 and OSPC2 cells, microarray and RT-qPCR analyses revealed the deregulation of several common and other unique subtype-specific FOXM1 putative targets involved in cell cycle, metastasis, DNA repair and drug response. CONCLUSIONS: FOXM1 is up-regulated in all three major EOCs subtypes, and is a prognostic biomarker and a potential combinatorial therapeutic target in platinum resistant disease, irrespective of tumor histology.

Antitumour activity of trabectedin in myelodysplastic/myeloproliferative neoplasms.

BACKGROUND: Juvenile myelomonocytic leukaemia (JMML) and chronic myelomonocytic leukaemia (CMML) are myelodysplastic myeloproliferative (MDS/MPN) neoplasms with unfavourable prognosis and without effective chemotherapy treatment. Trabectedin is a DNA minor groove binder acting as a modulator of transcription and interfering with DNA repair mechanisms; it causes selective depletion of cells of the myelomonocytic lineage. We hypothesised that trabectedin might have an antitumour effect on MDS/MPN. METHODS: Malignant CD14+ monocytes and CD34+ haematopoietic progenitor cells were isolated from peripheral blood/bone marrow mononuclear cells. The inhibition of CFU-GM colonies and the apoptotic effect on CD14+ and CD34+ induced by trabectedin were evaluated. Trabectedin's effects were also investigated in vitro on THP-1, and in vitro and in vivo on MV-4-11 cell lines. RESULTS: On CMML/JMML cells, obtained from 20 patients with CMML and 13 patients with JMML, trabectedin - at concentration pharmacologically reasonable, 1-5 nM - strongly induced apoptosis and inhibition of growth of haematopoietic progenitors (CFU-GM). In these leukaemic cells, trabectedin downregulated the expression of genes belonging to the Rho GTPases pathway (RAS superfamily) having a critical role in cell growth and cytoskeletal dynamics. Its selective activity on myelomonocytic malignant cells was confirmed also on in vitro THP-1 cell line and on in vitro and in vivo MV-4-11 cell line models. CONCLUSIONS: Trabectedin could be good candidate for clinical studies in JMML/CMML patients.

The bromodomain inhibitor OTX015 (MK-8628) exerts anti-tumor activity in triple-negative breast cancer models as single agent and in combination with everolimus.

Triple-negative breast cancer (TNBC) is an aggressive and heterogeneous subgroup of breast tumors clinically defined by the lack of estrogen, progesterone and HER2 receptors, limiting the use of the targeted therapies employed in other breast malignancies. Recent evidence indicates that c-MYC is a key driver of TNBC. The BET-bromodomain inhibitor OTX015 (MK-8628) has potent antiproliferative activity accompanied by c-MYC down-regulation in several tumor types, and has demonstrated synergism with the mTOR inhibitor everolimus in different models. The aim of this study was to evaluate the anti-tumor activity of OTX015 as single agent and in combination with everolimus in TNBC models. OTX015 was assayed in three human TNBC-derived cell lines, HCC1937, MDA-MB-231 and MDA-MB-468, all showing antiproliferative activity after 72 h (GI50 = 75-650 nM). This was accompanied by cell cycle arrest and decreased expression of cancer stem cells markers. However, c-MYC protein and mRNA levels were only down-regulated in MDA-MB-468 cells. Gene set enrichment analysis showed up-regulation of genes involved in epigenetic control of transcription, chromatin and the cell cycle, and down-regulation of stemness-related genes. In vitro, combination with everolimus was additive in HCC1937 and MDA-MB-231 cells, but antagonistic in MDA-MB-468 cells. In MDA-MB-231 murine xenografts, tumor mass was significantly (p < 0.05) reduced by OTX015 with respect to vehicle-treated animals (best T/C = 40.7%). Although everolimus alone was not active, the combination was more effective than OTX015 alone (best T/C = 20.7%). This work supports current clinical trials with OTX015 in TNBC (NCT02259114).

Promising in vivo efficacy of the BET bromodomain inhibitor OTX015/MK-8628 in malignant pleural mesothelioma xenografts.

It has recently been reported that a large proportion of human malignant pleural mesothelioma (MPM) cell lines and patient tissue samples present high expression of the c-MYC oncogene. This gene drives several tumorigenic processes and is overexpressed in many cancers. Although c-MYC is a strategic target to restrain cancer processes, no drugs acting as c-MYC inhibitors are available. The novel thienotriazolodiazepine small-molecule bromodomain inhibitor OTX015/MK-8628 has shown potent antiproliferative activity accompanied by c-MYC downregulation in several tumor types. This study was designed to evaluate the growth inhibitory effect of OTX015 on patient-derived MPM473, MPM487 and MPM60 mesothelioma cell lines and its antitumor activity in three patient-derived xenograft models, MPM473, MPM487 and MPM484, comparing it with cisplatin, gemcitabine and pemetrexed, three agents which are currently used to treat MPM in the clinic. OTX015 caused a significant delay in cell growth both in vitro and in vivo. It was the most effective drug in MPM473 xenografts and showed a similar level of activity as the most efficient treatment in the other two MPM models (gemcitabine in MPM487 and cisplatin in MPM484). In vitro studies showed that OTX015 downregulated c-MYC protein levels in both MPM473 and MPM487 cell lines. Our findings represent the first evidence of promising therapeutic activity of OTX015 in mesothelioma.

The HIV-protease inhibitor saquinavir reduces proliferation, invasion and clonogenicity in cervical cancer cell lines.

Innovative therapies in cervical cancer (CC) remain a priority. Recent data indicate that human immunodeficiency virus (HIV)-protease inhibitors used in highly active antiretroviral therapy can exert direct antitumor activities also in HIV-free preclinical and clinical models. The aim of the present study was to evaluate the antineoplastic effects of various HIV-protease inhibitors (indinavir, ritonavir and saquinavir) on primary and established CC cell lines. Two CC cell lines established in our laboratory and four commercially available CC cell lines were treated with indinavir, ritonavir and saquinavir at different concentrations and for different times. Proliferation, clonogenicity and radiosensitivity were evaluated by crystal violet staining. Proteasomal activities were assessed using a cell-based assay and immunoblotting. Cell cycle was analyzed by propidium iodide staining and flow cytometric analysis. Invasion was tested with Matrigel chambers. A t-test for paired samples was used for statistical analysis. In all cell lines, saquinavir was more effective than ritonavir in reducing cell proliferation and inhibiting proteasomal activities (P≤0.05). Conversely, indinavir exerted a negligible effect. The saquinavir concentrations required to modulate the proteasome activities were higher than those observed to be effective in inhibiting cell proliferation. In HeLa cells, saquinavir was strongly effective in inhibiting cell invasion and clonogenicity (P≤0.05) at concentrations much lower than those required to perturb proteasomal activities. Saquinavir did not contribute to increase the sensitivity of HeLa cells to X-rays. In conclusion, the present results demonstrate that saquinavir is able to significantly reduce cell proliferation, cell invasion and clonogenicity in a proteasome-independent manner in in vitro models of CC, and suggest that saquinavir could be a promising CC therapeutic agent.

Macrophages are essential for maintaining a M2 protective response early after ischemic brain injury.

Resident microglia and recruited macrophages are major contributors to the post-ischemic inflammatory response. Initially considered functionally homogeneous populations, data now suggest distinct but still controversial roles after brain injury. Using a model of conditional monocyte/macrophage depletion we studied the contribution of these myeloid cells to brain lesion progression after ischemia, and their influence on the ischemic inflammatory environment. Male CD11b-DTR transgenic mice, expressing the human diphtheria toxin receptor under the control of the CD11b promoter, were treated with diphtheria toxin to induce monocyte/macrophage depletion. Twenty four hours later the middle cerebral artery was permanently occluded. The ischemic lesion was measured 24h after injury. At the same time microglia and macrophage activation and polarization were assessed by quantitative immunohistochemistry and confocal microscopy for CD45high, CD11b, CD68, CD16/32, iNOS, Arg1, Ym1, and CD206, and gene expression was investigated on CD11b+ sorted cells. Depletion of monocytes/macrophages worsened the ischemic lesion within 24h after the ischemic insult. This effect was associated with higher M1/M2 polarization ratio in the ischemic lesion. Moreover, depletion increased the expression of M1 phenotypic markers on CD11b positive cells. Gene expression on CD11b+ sorted cells indicated a selective increase of iNOS and lower Arg1 mRNA expression than in non depleted mice. Depletion of monocytes/macrophages increases the ischemic lesion, an effect accompanied by an increase in the M1/M2 polarization ratio of microglia and macrophages in the ischemic area. Thus in ischemic injury recruited monocytes/macrophages may control an excessive M1 pro-inflammatory response, suggesting their ability to drive M2 protective polarization.

A new three dimensional biomimetic hydrogel to deliver factors secreted by human mesenchymal stem cells in spinal cord injury.

Stem cell therapy with human mesenchymal stem cells (hMSCs) represents a promising strategy in spinal cord injury (SCI). However, both systemic and parenchymal hMSCs administrations show significant drawbacks as a limited number and viability of stem cells in situ. Biomaterials able to encapsulate and sustain hMSCs represent a viable approach to overcome these limitations potentially improving the stem cell therapy. In this study, we evaluate a new agarose/carbomer based hydrogel which combines different strategies to optimize hMSCs viability, density and delivery of paracrine factors. Specifically, we evaluate a new loading procedure on a lyophilized scaffold (soaked up effect) that reduces mechanical stress in encapsulating hMSCs into the hydrogel. In addition, we combine arginine-glycine-aspartic acid (RGD) tripeptide and 3D extracellular matrix deposition to increase the capacity to attach and maintain healthy hMSCs within the hydrogel over time. Furthermore, the fluidic diffusion from the hydrogel toward the injury site is improved by using a cling film that oriented efficaciously the delivery of paracrine factors in vivo. Finally, we demonstrate that an improved combination as here proposed of hMSCs and biomimetic hydrogel is able to immunomodulate significantly the pro-inflammatory environment in a SCI mouse model, increasing M2 macrophagic population and promoting a pro-regenerative environment in situ.

Early modulation of pro-inflammatory microglia by minocycline loaded nanoparticles confers long lasting protection after spinal cord injury.

Many efforts have been performed in order to understand the role of recruited macrophages in the progression of spinal cord injury (SCI). Different studies revealed a pleiotropic effect played by these cells associated to distinct phenotypes (M1 and M2), showing a predictable spatial and temporal distribution in the injured site after SCI. Differently, the role of activated microglia in injury progression has been poorly investigated, mainly because of the challenges to target and selectively modulate them in situ. A delivery nanovector tool (poly-ε-caprolactone-based nanoparticles) able to selectively treat/target microglia has been developed and used here to clarify the temporal and spatial involvement of the pro-inflammatory response associated to microglial cells in SCI. We show that a treatment with nanoparticles loaded with minocycline, the latter a well-known anti-inflammatory drug, when administered acutely in a SCI mouse model is able to efficiently modulate the resident microglial cells reducing the pro-inflammatory response, maintaining a pro-regenerative milieu and ameliorating the behavioral outcome up to 63 days post injury. Furthermore, by using this selective delivery tool we demonstrate a mechanistic link between early microglia activation and M1 macrophages recruitment to the injured site via CCL2 chemokine, revealing a detrimental contribution of pro-inflammatory macrophages to injury progression after SCI.

3D Silicon Microstructures: A New Tool for Evaluating Biological Aggressiveness of Tumor Cells.

In this work, silicon micromachined structures (SMS), consisting of arrays of 3- µ m-thick silicon walls separated by 50- µm-deep, 5- µ m-wide gaps, were applied to investigate the behavior of eight tumor cell lines, with different origins and biological aggressiveness, in a three-dimensional (3D) microenvironment. Several cell culture experiments were performed on 3D-SMS and cells grown on silicon were stained for fluorescence microscopy analyses. Most of the tumor cell lines recognized in the literature as highly aggressive (OVCAR-5, A375, MDA-MB-231, and RPMI-7951) exhibited a great ability to enter and colonize the narrow deep gaps of the SMS, whereas less aggressive cell lines (OVCAR-3, Capan-1, MCF7, and NCI-H2126) demonstrated less penetration capability and tended to remain on top of the SMS. Quantitative image analyses of several fluorescence microscopy fields of silicon samples were performed for automatic cell recognition and count, in order to quantify the fraction of cells inside the gaps, with respect to the total number of cells in the examined field. Our results show that higher fractions of cells in the gaps are obtained with more aggressive cell lines, thus supporting in a quantitative way the observation that the behavior of tumor cells on the 3D-SMS depends on their aggressiveness level.

Antiangiogenic activity of trabectedin in myxoid liposarcoma: involvement of host TIMP-1 and TIMP-2 and tumor thrombospondin-1.

Trabectedin is a marine natural product, approved in Europe for the treatment of soft tissue sarcoma and relapsed ovarian cancer. Clinical and experimental evidence indicates that trabectedin is particularly effective against myxoid liposarcomas where response is associated to regression of capillary networks. Here, we investigated the mechanism of the antiangiogenic activity of trabectedin in myxoid liposarcomas. Trabectedin directly targeted endothelial cells, impairing functions relying on extracellular matrix remodeling (invasion and branching morphogenesis) through the upregulation of the inhibitors of matrix metalloproteinases TIMP-1 and TIMP-2. Increased TIMPs synthesis by the tumor microenvironment following trabectedin treatment was confirmed in xenograft models of myxoid liposarcoma. In addition, trabectedin upregulated tumor cell expression of the endogenous inhibitor thrombospondin-1 (TSP-1, a key regulator of angiogenesis-dependent dormancy in sarcoma), in in vivo models of myxoid liposarcomas, in vitro cell lines and primary cell cultures from patients' myxoid liposarcomas. Chromatin Immunoprecipitation analysis showed that trabectedin displaced the master regulator of adipogenesis C/EBPß from the TSP-1 promoter, indicating an association between the up-regulation of TSP-1 and induction of adipocytic differentiation program by trabectedin. We conclude that trabectedin inhibits angiogenesis through multiple mechanisms, including directly affecting endothelial cells in the tumor microenvironment--with a potentially widespread activity--and targeting tumor cells' angiogenic activity, linked to a tumor-specific molecular alteration.

Microtubule-associated protein/microtubule affinity-regulating kinase 4 (MARK4) plays a role in cell cycle progression and cytoskeletal dynamics.

MARK4 is a serine-threonine kinase that phosphorylates MAP proteins, increasing microtubule dynamics. MARK4 differs from the other members of the MARK family for encoding two isoforms (MARK4L and MARK4S), differentially expressed in the nervous system, and for the peculiar localisation at the centrosome and the midbody. By cytofluorimetric analysis we showed that MARK4 is expressed throughout the cell cycle and preferentially activated during mitosis. Depletion of MARK4S affected the morphology and proliferation of fibroblasts and glioma cells, as the percentages of cells in S and G2/M phases were reduced and the percentage of cells in G1 was increased. In MARK4S silenced cells, centrosomes were duplicated and positioned apically to the nucleus, indicating that the centrosome cycle was altered and the cells arrested in G1 phase. Overexpression of MARK4L or MARK4S reduced the density of the microtubule network, confirming microtubules as the main target of MARK4, and revealed a novel co-localisation of MARK4 and vimentin. Taken together, our data confirm that MARK4 is a key component in the regulation of microtubule dynamics and highlight its major role in cell cycle progression, particularly at the G1/S transition. The co-localisation of vimentin and MARK4L suggests that MARK4 has a wide-ranging influence on cytoskeleton.

Resistance to minor groove binders.

In this paper multiple resistance mechanisms to minor groove binders (MGBs) are overviewed. MGBs with antitumor properties are natural products or their derivatives and, as expected, they are all substrates of P-glycoprotein (P-gp). However, a moderate expression of P-gp does not appear to reduce the sensitivity to trabectedin, the only MGB so far approved for clinical use. Resistance to this drug is often related to transcriptional mechanisms and to DNA repair pathways, particularly defects in transcription-coupled nucleotide excision repair (TC-NER). Therefore tumors resistant to trabectedin may become hypersensitive to UV rays and other DNA damaging agents acting in the major groove, such as Platinum (Pt) complexes. If this is confirmed in clinic, that will provide the rationale to combine trabectedin sequentially with Pt derivates.

Comparative analysis of multilineage properties of mesenchymal stromal cells derived from fetal sources shows an advantage of mesenchymal stromal cells isolated from cord blood in chondrogenic differentiation potential.

BACKGROUND AIMS: Cord blood (CB) and amniotic fluid (AF) could represent new and attractive mesenchymal stromal cell (MSC) sources, but their potential therapeutic applications are still limited by lack of standardized protocols for isolation and differentiation. In particular, chondrogenic differentiation has never been deeply investigated. METHODS: MSCs were obtained from CB and AF samples collected during cesarean sections at term and compared for their biological and differentiation properties, with particular interest in cartilage differentiation, in which quantitative real-time polymerase chain reaction and immunohistochemical analyses were performed to evaluate the expression of type 2 collagen, type 10 collagen, SRY-box9 and aggrecan. RESULTS: We were able to isolate MSCs from 12 of 30 (40%) and 5 of 20 (25%) CB and AF units, respectively. Fluorescence in situ hybridization analysis indicated the fetal origin of isolated MSC strains. Both populations expressed mesenchymal but not endothelial and hematopoietic markers, even though we observed a lower expression of human leukocyte antigen (HLA) I in CB-MSCs. No differences in proliferation rate and cell cycle analysis could be detected. After osteogenic induction, both populations showed matrix mineralization and typical marker expression. Under chondrogenic conditions, pellets derived from CB-MSCs, in contrast with AF-MSCs pellets, were significantly larger, showed cartilage-like morphology and resulted positive for chondrocyte-associated markers, such as type 2 collagen, type 10 collagen, SRY-box9 and aggrecan. CONCLUSIONS: Our results show that CB-MSCs and AF-MSCs collected at term differ from each other in their biological and differentiation properties. In particular, only CB-MSCs showed a clear chondrogenic potential and thus could represent an ideal candidate for cartilage-tissue engineering.