Abstract-To determine whether the peripheral blood in humans contains a population of multipotent progenitor cells (MPCs), products of leukapheresis were obtained from healthy donor volunteers following the administration of granulocyte colony-stimulating factor. Small clusters of adherent proliferating cells were collected, and these cells continued to divide up to 40 population doublings without reaching replicative senescence and growth arrest. MPCs were positive for the transcription factors Nanog, Oct3/4, Sox2, c-Myc, and Klf4 and expressed several antigens characteristic of mesenchymal stem cells. However, they were negative for markers of hematopoietic stem/progenitor cells and bone marrow cell lineages. MPCs had a cloning efficiency of Ϸ3%, and following their expansion, retained a highly immature phenotype. Under permissive culture conditions, MPCs differentiated into neurons, glial cells, hepatocytes, cardiomyocytes, endothelial cells, and osteoblasts. Moreover, the gene expression profile of MPCs partially overlapped with that of neural and embryonic stem cells, further demonstrating their primitive, uncommitted phenotype. Following subcutaneous transplantation in nonimmunosuppressed mice, MPCs migrated to distant organs and integrated structurally and functionally within the new tissue, acquiring the identity of resident parenchymal cells. In conclusion, undifferentiated cells with properties of embryonic stem cells can be isolated and expanded from human peripheral blood after granulocyte colony-stimulating factor administration. This cell pool may constitute a unique source of autologous cells with critical clinical import.

The In-Check System is based on a miniaturized silicon lab-on-chip (LoC) where the Polymerase Chain reactor lives together with a customizable microarray module for running a seamless nucleic acid test [1]. This device is designed for accurate temperature performances control, such as accuracy and heating rate provided by both a sophisticated chip calibration process and a precise control by the Temperature Control System (TCS). In addition, the device, is optimized for a microarray fluorescence reading operation by an external instrument, the optical reader (OR). Finally, it is based on microfluidic features that enable to load the chip and fill the reaction chambers without the risk of bubble formation or leaks.In this manuscript are reported the experimental results for the detection of human betaglobine gene (HBB) directly from human cells in less than 2 hours in a silicon reactor. The sample preparation process was entirely performed in one single step into the silicon reactor. It was fully characterized by RT-qPCR. We performed also a comparison study showing higher performances in the LoC silicon reactor than the standard tube. Moreover, the DNA extracted was amplified by PCR, and the resulting product hybridized on the microarray. All the results suggest that the hybridization reactions performed on the silicon LoC can be used to exploit the discriminatory power of microarrays for a specific gene detection.

cCalpains regulate a wide spectrum of biological functions, including migration, adhesion, apoptosis, secretion, and autophagy, through the modulating cleavage of specific substrates. Ubiquitous microcalpain (-calpain) and millicalpain (m-calpain) are heterodimers composed of catalytic subunits encoded, respectively, by CAPN1 and CAPN2 and a regulatory subunit encoded by CAPNS1. Here we show that calpain is required for the stability of the deubiquitinating enzyme USP1 in several cell lines. USP1 modulates DNA replication polymerase choice and repair by deubiquitinating PCNA. The ubiquitinated form of the USP1 substrate PCNA is stabilized in CAPNS1-depleted U2OS cells and mouse embryonic fibroblasts (MEFs), favoring polymeraseloading on chromatin and increased mutagenesis. USP1 degradation directed by the cell cycle regulator APC/C cdh1 , which marks USP1 for destruction in the G 1 phase, is upregulated in CAPNS1-depleted cells. USP1 stability can be rescued upon forced expression of calpain-activated Cdk5/p25, previously reported as a cdh1 repressor. These data suggest that calpain stabilizes USP1 by activating Cdk5, which in turn inhibits cdh1 and, consequently, USP1 degradation. Altogether these findings point to a connection between the calpain system and the ubiquitin pathway in the regulation of DNA damage response and place calpain at the interface between cell cycle modulation and DNA repair. Calpains regulate a wide spectrum of biological functions, including migration, adhesion, apoptosis, secretion, and autophagy through the modulated cleavage of specific substrates (reviewed in references 1-3). Ubiquitous microcalpain (-calpain) and millicalpain (m-calpain) are heterodimers composed of a catalytic subunit encoded, respectively, by CAPN1 and CAPN2 and a regulatory subunit encoded by CAPNS1. Both -calpain and mcalpain are negatively modulated by calpastatin. By a proteomic approach, we identified USP1 deubiquitinase as a CAPNS1-interacting protein. USP1 is a key modulator of DNA repair, partly through deubiquitination of its known targets FANCD2 (4, 5) and PCNA (6). Usp1 knockout (KO) mice have a severe phenotype and die soon after birth (7). Usp1 Ϫ/Ϫ cells are defective in FANCD2 focus formation and are hypersensitive to DNA damage (8). PCNA ubiquitination is higher in USP1-depleted cells than in control cells, thus leading to recruitment of error-prone, translesion DNA synthesis (TLS) polymerases and the consequent increase in mutation rate (6). USP1 promotes inhibitor of DNA binding (ID) protein stability and stem cell-like characteristics in osteosarcoma and is required for normal skeletogenesis (9). Interestingly, mice lacking CAPNS1 in cells of the osteoblast lineage are defective in bone development and remodeling in vivo (10).UV light irradiation activates hUSP1 autocleavage at glycines 670 and 671, inducing its subsequent proteasomal degradation (6). USP1 mutants with mutation in the catalytic cysteine 90 or in the autocleavage sites are more stable but can still be degraded in the cell (5), s...

Mesenchymal stem/stromal cells (MSCs) are the precursors of various cell types that compose both normal and cancer tissue microenvironments. In order to support the widely diversified parenchymal cells and tissue organization, MSCs are characterized by a large degree of heterogeneity, although available analyses of molecular and transcriptional data do not provide clear evidence. We have isolated MSCs from high-grade serous ovarian cancers (HG-SOCs) and various normal tissues (N-MSCs), demonstrated their normal genotype and analyzed their transcriptional activity with respect to the large comprehensive FANTOM5 sample dataset. Our integrative analysis conducted against the extensive panel of primary cells and tissues of the FANTOM5 project allowed us to mark the HG-SOC-MSCs CAGE-seq transcriptional heterogeneity and to identify a cell-type-specific transcriptional activity showing a significant relationship with primary mesothelial cells. Our analysis shows that MSCs isolated from different tissues are highly heterogeneous. The mesothelial-related gene signature identified in this study supports the hypothesis that HG-SOC-MSCs are bona fide representatives of the ovarian district. This finding indicates that HG-SOC-MSCs could actually derive from the coelomic mesothelium, suggesting that they might be linked to the epithelial tumor through common embryological precursors. STEM CELLS 2014;32:2998-3011

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