Fast single-cell biochemistry: theory, open source microscopy and applications

Fluorescence lifetime sensing enables researchers to probe the physicochemical environment of a fluorophore providing a window through which we can observe the complex molecular make-up of the cell. Fluorescence lifetime imaging microscopy (FLIM) quantifies and maps cell biochemistry, a complex ensemble of dynamic processes. Unfortunately, typical high-resolution FLIM systems exhibit rather limited acquisition speeds, often insufficient to capture the time evolution of biochemical processes in living cells. Here, we describe the theoretical background that justifies the developments of high-speed single photon counting systems. We show that systems with low dead-times not only result in faster acquisition throughputs but also improved dynamic range and spatial resolution. We also share the implementation of hardware and software as an open platform, show applications of fast FLIM biochemical imaging on living cells and discuss strategies to balance precision and accuracy in FLIM. The recent innovations and commercialisation of fast time-domain FLIM systems are likely to popularise FLIM within the biomedical community, to impact biomedical research positively and to foster the adoption of other FLIM techniques as well. While supporting and indeed pursuing these developments, with this work we also aim to warn the community about the possible shortcomings of fast single photon counting techniques and to highlight strategies to acquire data of high quality.We acknowledge funding from the Medical Research Council core grants (MC_UU_12022/1 and MC_UU_12022/8) to ARV

Contributions to Drug Resistance in Glioblastoma Derived from Malignant Cells in the Sub-Ependymal Zone

Glioblastoma (GB), the most common and aggressive adult brain tumor, is characterized by extreme phenotypic diversity and treatment failure. Through fluorescence-guided resection, we identified fluorescent tissue in the sub-ependymal zone (SEZ) of GB patients. Histological analysis and genomic characterization revealed that the SEZ harbors malignant cells with tumor-initiating capacity, analogous to cells isolated from the fluorescent tumor mass (T). We observed resistance to supra-maximal chemotherapy doses along with differential patterns of drug response between T and SEZ in the same tumor. Our results reveal novel insights into GB growth dynamics, with implications for understanding and limiting treatment resistance

Dedifferentiation of Foetal CNS Stem Cells to Mesendoderm-Like Cells through an EMT Process

Tissue-specific stem cells are considered to have a limited differentiation potential. Recently, this notion was challenged by reports that showed a broader differentiation potential of neural stem cells, in vitro and in vivo, although the molecular mechanisms that regulate plasticity of neural stem cells are unknown. Here, we report that neural stem cells derived from mouse embryonic cortex respond to Lif and serum in vitro and undergo epithelial to mesenchymal transition (EMT)-mediated dedifferentiation process within 48 h, together with transient upregulation of pluripotency markers and, more notably, upregulation of mesendoderm genes, Brachyury (T) and Sox17. These induced putative mesendoderm cells were injected into early gastrulating chick embryos, which revealed that they integrated more efficiently into mesoderm and endoderm lineages compared to non-induced cells. We also found that TGFβ and Jak/Stat pathways are necessary but not sufficient for the induction of mesendodermal phenotype in neural stem cells. These results provide insights into the regulation of plasticity of neural stem cells through EMT. Dissecting the regulatory pathways involved in these processes may help to gain control over cell fate decisions

Adulten Stammzellen aus dem Knochemark: Charakterizierung und ihre Applikation für die Zellen Therapie

Embryonale Stammzellen sind aufgrund ihrer hohen Plastizität ein sehr viel versprechendes therapeutisches Mittel für die Regenerative Medizin. Ethische und beträchtliche klinische Probleme schränken ihren Einsatz jedoch ein. Im Gegensatz hierzu zeigen adulte Stammzellen positivere Zukunftsaussichten für die Regenerative Medizin auf. Das multilineage Potential, die einfache Isolierung und die Tatsache, dass die Transplantation dieser Zellen nicht zu schwerwiegenden klinischen Problemen, wie z.B. Tumorbildung, führt, sind dabei von Vorteil. In den vergangenen sechs Jahren gab es zudem vermehrt Daten über Knochmarksstammzellen und deren hohe Plastizität. In diesem Zusammenhang beschäftigte sich unsere Arbeit vorrangig mit der Verbesserung der Kultivierungsbedingungen dieser Zellen. Ziel war es, eine Stammzellkultur mit hoher Plastizität zu generieren. Hierzu wurden Knochenmarksstammzellen in Medium mit verschiedenen Wachstumsfaktoren, einschließlich LIF, kultiviert. Durch in vivo Aggregation dieser Zellen mit Morulae wurde deren hohe Plastizität bestätigt. Die Vermehrung der multipotenten Knochenmarksstammzellen sollte mit Hilfe transgener Mäuse umgesetzt werden, die GFP unter einem Oct4-Promotor exprimieren. Bereits nach kurzer Kultivierung der isolierten Knochenmarkszellen konnte man GFP-positive Zellcluster finden. Mit Hilfe der Real-time-PCR wurde in den Knochenmarksstammzellen eine 30mal geringere Oct4- und eine 10mal geringere Nanog-Expression als in embryonalen Stammzellen festgestellt. Im Vergleich zu anderen Geweben sind die Oct4- und die Nanog-Expression aber dennoch sehr hoch. Deshalb wurden die Stammzellcluster isoliert und zum Vergleich sowohl in Nanog-haltigem als auch in FGF2-haltigem Medium kultiviert, um sie in vitro zu vermehren. Die Zugabe des TAT-Nanog-Proteins in das Medium steigerte die Zellproliferation bedeutend. Obwohl hier eine hohe Sox2-Expression zu beobachten ist, trifft dies jedoch nicht auf die Oct4-Expression zu. Die Zugabe von FGF2 hingegen hatte einen unmittelbaren Effekt auf die Induktion der Oct4-Expression in den Stammzellkulturen, der auch über mehrere Passagen anhielt

Yönlendirilmiş doku mühendisliği amaçlı kollajen taşıyıcılar

M.S. - Master of Scienc

Expression of pluripotency associated proteins in serum-free cultured neurospheres and 48 h serum and Lif induced neurospheres.

<p>(<b>A</b>): Morphology in serum-free (upper panel) and 48 h serum/Lif (lower panel) conditions. (<b>B</b>): Transcriptional profile of pluripotency associated factors Oct4, Sox2, c-Myc, Klf4 and Nanog of serum-free cultured neurospheres and 48 h treated neurospheres. Data is expressed as ΔΔCT and normalized to ES cells. (<b>C</b>): Immunostaining for Oct4, Nanog, Sox2, SSEA1 and alkaline phosphatase. Abbreviations: AP, alkaline phosphatase; SSEA1, stage specific embryonic antigen 1. Scale bars: 50 µm.</p

<i>In vitro</i> differentiation of neurospheres in serum and Lif conditions.

<p>(<b>A</b>): Immunostaining for Brachyury (T), Sox17, Nanog and Oct4 at 5 days after induction in serum and Lif conditions (<b>B</b>): At 10 days post induction with serum and Lif, cells exhibit very heterogeneous morphologies, indicating the presence of different cell types. (<b>C</b>): Immunostaining for GFAP and αSMA in 10 day induced cultures. Abbreviations: GFAP, glial fibrillary acidic protein; αSMA, alpha smooth muscle actin. Scale bars: 50 µm.</p

<i>In vivo</i> differentiation of 48 h induced neural stem cells.

<p>Staining of chick embryo paraffin sections for mesenchymal marker N-cadherin, tight junction marker ZO-1, endoderm marker Sox17, and epithelial marker E-cadherin. Although some of the labelled cells express E-cadherin, the staining pattern suggest the cells do not achieve a complete integration to ectoderm lineage. However, high integration towards mesoderm and endoderm lineages is confirmed by N-cadherin, ZO-1 and Sox17 stainings. Although induced (green) cells show higher efficiency of integration, both induced (green) and non-induced (red) cells once incorporated into these tissues express respective lineage markers and acquire similar morphological characteristics to their neighbouring host cells.</p

EMT and mesendoderm markers upregulated in neurospheres after 48 h induction with serum and Lif.

<p>(<b>A</b>): Immunostaining for Slug, E-cadherins and N-cadherins (<b>B</b>): Immunostaining for mesendoderm markers Sox17 and Brachyury (T). (<b>C</b>): Expression profile of Slug, N-cadherins, E-cadherins, Sox17 and Brachyury of 48 h serum and Lif induced neurospheres measured by QPCR relative to neurospheres cultured in standard neural stem cell media. Abbreviations: Ncad, N-cadherins; Ecad, E-cadherins. Scale bars: 50 µm.</p

Schematic diagram depicting our interpretation of the results.

<p>Neural stem cells derived from later stages of development have been shown to reprogram to iPS cells via overexpression of <i>Oct4</i> transcription factor (Kim et al., 2009). Signalling alone (BMPs and bFGF), however, can induce dedifferentiation of neural stem cells into a neural crest phenotype (Sailer et al., 2005). TGFβ and Jak/Stat pathways can induce a further dedifferentiation to mesendoderm-like phenotype providing evidence for extracellular signaling regulated cell plasticity of neural stem cells (this work, red arrow).</p