Mesenchymal stem cells (MSCs) have emerged as important tools for cell therapy; therefore, identification of factors capable of governing their ex vivo expansion become essential. In this study we demonstrate that human adipose-derived stem cells (ASCs) express all components of the bone morphogenetic protein (BMP)/BMP receptor signaling pathway and respond to BMP4 inducing upregulated expression of its specific target genes Id1-Id4. Moreover, ASCs grown in a medium reduced in serum produce endogenous BMP4 that could affect autocrinely ASC growth. On the contrary, dorsomorphin, an inhibitor of BMP signaling pathway, decreases cell numbers yielded from ASC cultures in correlation with increased apoptosis and decreased cycling cells. Therefore, BMP4 emerges as a possible factor for ex vivo expanding human ASCs. Our results demonstrate that, as other morphogens, BMP4 effects on human MSCs are dose dependent. High doses significantly increased apoptosis and drastically reduced cell proliferation, whereas low doses of BMP4 (0.01-0.1 ng/mL) significantly increase culture cell content, reduce the number of apoptotic cells, and increase that of cycling cells. Further, treatment of human ASCs with low doses of BMP4 does not modify expression of Nanog and Oct4, two transcription factors involved in self-renewal and pluripotency of stem cells or avoid their osteogenic or osteoblastic differentiation capacities when cultured in adequate inducing media, as shown by the induction of specific gene expression (CEBP, PPARγ, and RUNX2). Our results therefore support BMP4 as a promising factor for expanding human adipose tissue-derived MSCs maintaining their properties of stemness and multipotency.

KRAS mutations are responsible for resistance to anti-epidermal growth factor receptor (EGFR) therapy in colorectal cancer patients. These mutations sometimes appear once treatment has started. Detection of KRAS mutations in circulating cell-free DNA in plasma (“liquid biopsy”) by droplet digital PCR (ddPCR) has emerged as a very sensitive and promising alternative to serial biopsies for disease monitoring. In this study, KRAS G12V mutation was analyzed by ddPCR in plasma DNA from 10 colorectal cancer patients and compared to six healthy donors. The percentage of KRAS G12V mutation relative to wild-type sequences in tumor-derived DNA was also determined. KRAS G12V mutation circulating in plasma was detected in 9 of 10 colorectal cancer patients whose tumors were also mutated. Colorectal cancer patients had 35.62 copies of mutated KRAS/mL plasma, whereas in healthy controls only residual copies were found (0.62 copies/mL, p = 0.0066). Interestingly, patients with metastatic disease showed a significantly higher number of mutant copies than M0 patients (126.25 versus 9.37 copies/mL, p = 0.0286). Wild-type KRAS was also significantly elevated in colorectal cancer patients compared to healthy controls (7718.8 versus 481.25 copies/mL, p = 0.0002). In conclusion, KRAS G12V mutation is detectable in plasma of colorectal cancer patients by ddPCR and could be used as a non-invasive biomarker.

Background. Hemophilia is a rare recessive X-linked disease characterized by a deficiency of coagulation factor VIII or factor IX. Its current treatment is merely palliative. Advanced therapies are likely to become the treatment of choice for the disease as they could provide a curative treatment.Methods. The present study looks into the use of a safe non-viral transfection method based on nucleofection to express and secrete human clotting factor IX (hFIX) where human adipose tissue derived mesenchymal stem cells were used as target cells in vitro studies and NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice were used to analyze factor IX expression in vivo studies. Previously, acute liver injury was induced by an injected intraperitoneal dose of 500 mg/kg body weight of acetaminophen.Results. Nucleofection showed a percentage of positive cells ranging between 30.7% and 41.9% and a cell viability rate of 29.8%, and cells were shown to secrete amounts of hFIX between 36.8 and 71.9 ng/mL. hFIX levels in the blood of NSG mice injected with ASCs transfected with this vector, were 2.7 ng/mL 48 h after injection. Expression and secretion of hFIX were achieved both in vitro cell culture media and in vivo in the plasma of mice treated with the transfected ASCs. Such cells are capable of eventually migrating to a previously damaged target tissue (the liver) where they secrete hFIX, releasing it to the bloodstream over a period of at least five days from administration.Conclusions. The results obtained in the present study may form a preliminary basis for the establishment of a future ex vivo non-viral gene/cellular safe therapy protocol that may eventually contribute to advancing the treatment of hemophilia.