Sorting living mesenchymal stem cells using a TWIST1 RNA-based probe depends on incubation time and uptake capacity

Bone marrow derived mesenchymal stromal cells (BMSCs) are multipotent progenitors of particular interest for cell-based tissue engineering therapies. However, one disadvantage that limit their clinical use is their heterogeneity. In the last decades a great effort was made to select BMSC subpopulations based on cell surface markers, however there is still no general consensus on which markers to use to obtain the best BMSCs for tissue regeneration. Looking for alternatives we decided to focus on a probe-based method to detect intracellular mRNA in living cells, the SmartFlare technology. This technology does not require fixation of the cells and allows us to sort living cells based on gene expression into functionally different populations. However, since the technology is available it is debated whether the probes specifically recognize their target mRNAs. We validated the TWIST1 probe and demonstrated that it specifically recognizes TWIST1 in BMSCs. However, differences in probe concentration, incubation time and cellular uptake can strongly influence signal specificity. In addition we found that TWIST1high expressing cells have an increased expansion rate compared to TWIST1low expressing cells derivedfrom the same initial population of BMSCs. The SmartFlare probes recognize their target gene, however for each probe and cell type validation of the protocol is necessary

Population Pharmacokinetic Model and Pharmacokinetic Target Attainment of Micafungin in Intensive Care Unit Patients

_Objective:_ To study the pharmacokinetics of micafungin in intensive care patients and assess pharmacokinetic (PK) target attainment for various dosing strateg

Contraction pressure analysis using optical imaging in normal and MYBPC3-mutated hiPSC-derived cardiomyocytes grown on matrices with tunable stiffness

Current in vivo disease models and analysis methods for cardiac drug development have been insufficient in providing accurate and reliable predictions of drug efficacy and safety. Here, we propose a custom optical flow-based analysis method to quantitatively measure recordings of contracting cardiomyocytes on polydimethylsiloxane (PDMS), compatible with medium-throughput systems. Movement of the PDMS was examined by covalently bound fluorescent beads on the PDMS surface, differences caused by increased substrate stiffness were compared, and cells were stimulated with β-agonist. We further validated the system using cardiomyocytes treated with endothelin-1 and compared their contractions against control and cells incubated with receptor antagonist bosentan. After validation we examined two MYBPC3-mutant patient-derived cell lines. Recordings showed that higher substrate stiffness resulted in higher contractile pressure, while beating frequency remained similar to control. β-agonist stimulation resulted in both higher beating frequency as well as higher pressure values during contraction and relaxation. Cells treated with endothelin-1 showed an increased beating frequency, but a lower contraction pressure. Cells treated with both endothelin-1 and bosentan remained at control level of beating frequency and pressure. Lastly, both MYBPC3-mutant lines showed a higher beating frequency and lower contraction pressure. Our validated method is capable of automatically quantifying contraction of hiPSC-derived cardiomyocytes on a PDMS substrate of known shear modulus, returning an absolute value. Our method could have major benefits in a medium-throughput setting.</p

Human mesenchymal stromal cells in adhesion to cell-derived extracellular matrix and titanium: Comparative kinome profile analysis

The extracellular matrix (ECM) physically supports cells and influences stem cell behaviour, modulating kinase-mediated signalling cascades. Cell-derived ECMs have emerged in bone regeneration as they reproduce physiological tissue-architecture and ameliorate mesenchymal stromal cell (MSC) properties. Titanium scaffolds show good mechanical properties, facilitate cell adhesion, and have been routinely used for bone tissue engineering (BTE). We analyzed the kinomic signature of human MSCs in adhesion to an osteopromotive osteoblast-derived ECM, and compared it to MSCs on titanium. PamChip kinase-array analysis revealed 63 phosphorylated peptides on ECM and 59 on titanium, with MSCs on ECM exhibiting significantly higher kinase activity than on titanium. MSCs on the two substrates showed overlapping kinome profiles, with activation of similar signalling pathways (FAK, ERK, and PI3K signalling). Inhibition of PI3K signalling in cells significantly reduced adhesion to ECM and increased the number of nonadherent cells on both substrates. In summary, this study comprehensively characterized the kinase activity in MSCs on cell-derived ECM and titanium, highlighting the role of PI3K signalling in kinomic changes regulating osteoblast viability and adhesion. Kinome profile analysis represents a powerful tool to select pathways to better understand cell behaviour. Osteoblast-derived ECM could be further investigated as titanium scaffold-coating to improve BTE

Hypothermic Machine Perfusion in Liver Transplantation - A Randomized Trial

BACKGROUND Transplantation of livers obtained from donors after circulatory death is associated with an increased risk of nonanastomotic biliary strictures. Hypothermic oxygenated machine perfusion of livers may reduce the incidence of biliary complications, but data from prospective, controlled studies are limited. METHODS In this multicenter, controlled trial, we randomly assigned patients who were undergoing transplantation of a liver obtained from a donor after circulatory death to receive that liver either after hypothermic oxygenated machine perfusion (machine-perfusion group) or after conventional static cold storage alone (control group). The primary end point was the incidence of nonanastomotic biliary strictures within 6 months after transplantation. Secondary end points included other graft-related and general complications. RESULTS A total of 160 patients were enrolled, of whom 78 received a machine-perfused liver and 78 received a liver after static cold storage only (4 patients did not receive a liver in this trial). Nonanastomotic biliary strictures occurred in 6% of the patients in the machine-perfusion group and in 18% of those in the control group (risk ratio, 0.36; 95% confidence interval [CI], 0.14 to 0.94; P=0.03). Postreperfusion syndrome occurred in 12% of the recipients of a machine-perfused liver and in 27% of those in the control group (risk ratio, 0.43; 95% CI, 0.20 to 0.91). Early allograft dysfunction occurred in 26% of the machine-perfused livers, as compared with 40% of control livers (risk ratio, 0.61; 95% CI, 0.39 to 0.96). The cumulative number of treatments for nonanastomotic biliary strictures was lower by a factor of almost 4 after machine perfusion, as compared with control. The incidence of adverse events was similar in the two groups. CONCLUSIONS Hypothermic oxygenated machine perfusion led to a lower risk of nonanastomotic biliary strictures following the transplantation of livers obtained from donors after circulatory death than conventional static cold storage

Adjuvant hyperthermic intraperitoneal chemotherapy (HIPEC) in patients with colon cancer at high risk of peritoneal carcinomatosis; the COLOPEC randomized multicentre trial

Background: The peritoneum is the second most common site of recurrence in colorectal cancer. Early detection of peritoneal carcinomatosis (PC) by imaging is difficult. Patients eventually presenting with clinically apparent PC have a poor prognosis. Median survival is only about five months if untreated and the benefit of palliative systemic chemotherapy is limited. Only a quarter of patients are eligible for curative treatment, consisting of cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CR/HIPEC). However, the effectiveness depends highly on the extent of disease and the treatment is associated with a considerable complication rate. Methods/Design: The aim of this study is to determine the effectiveness of adjuvant HIPEC in preventing the development of PC in patients with colon cancer at high risk of peritoneal recurrence. This study will be performed in the nine Dutch HIPEC centres, starting in April 2015. Eligible for inclusion are patients who underwent curative resection for T4 or intra-abdominally perforated cM0 stage colon cancer. After resection of the primary tumour, 176 patients will be randomized to adjuvant HIPEC followed by routine adjuvant systemic chemotherapy in the experimental arm, or to systemic chemotherapy only in the control arm. Adjuvant HIPEC will be performed simultaneously or shortly after the primary resection. Oxaliplatin will be used as chemotherapeutic agent, for 30 min at 42-43 °C. Just before HIPEC, 5-fluorouracil and leucovorin will be administered intravenously. Primary endpoint is peritoneal disease-free survival at 18 months. Diagnostic laparoscopy will be performed routinely after 18 months postoperatively in both arms of the study in patients without evidence of disease based on routine follow-up using CT imaging and CEA. Discussion: Adjuvant HIPEC is assumed to reduce the expected 25 % absolute risk of PC in patients with T4 or perforated colon cancer to a risk of 10 %. This reduction is likely to translate into a prolonged overall survival. Trial registration number: NCT02231086 (Clinicaltrials.gov)

Spitzer Observations of G Dwarfs in the Pleiades: Circumstellar Debris Disks at 100 Myr Age

Fluxes and upper limits in the wavelength range from 3.6 to 70 microns from the Spitzer Space Telescope are provided for twenty solar-mass Pleiades members. One of these stars shows a probable mid-IR excess and two others have possible excesses, presumably due to circumstellar debris disks. For the star with the largest, most secure excess flux at MIPS wavelengths, HII1101, we derive Log(L[dust]/L[Sun]) ~ -3.8 and an estimated debris disk mass of 4.2 x 10^-5 M(Earth) for an assumed uniform dust grain size of 10 microns If the stars with detected excesses are interpreted as stars with relatively recent, large collision events producing a transient excess of small dust particles, the frequency of such disk transients is about ~ 10 % for our ~ 100 Myr, Pleiades G dwarf sample. For the stars without detected 24-70 micron excesses, the upper limits to their fluxes correspond to approximate 3 sigma upper limits to their disk masses of 6 x 10^-6 M(Earth) using the MIPS 24 micron upper limit, or 2 x 10^-4 M(Earth) using the MIPS 70 micron limit. These upper limit disk masses (for "warm" and "cold" dust, respectively) are roughly consistent, but somewhat lower than, predictions of a heuristic model for the evolution of an "average" solar-mass star's debris disk based on extrapolation backwards in time from current properties of the Sun's Kuiper belt.Comment: 32 postscript pages including 8 figues and 3 tables. To appear in the Astronomical Journa