ABSTRACT
Haematopoietic stem cells (HSCs) have been extensively characterized regarding in vivo engraftment, surface epitopes and genetic regulation. However, little is known about the homing of these rare cells, and their intrinsic motility and membrane deformation capacity. We used high-speed optical-sectioning microscopy and inverted fluorescent videomicroscopy to study highly purified murine lineage-negative, rhodamine-low, Hoechst-low HSCs over time under various in vitro conditions. We discovered extremely rapid motility, directed migration to stromal cells and marked membrane modulation. High resolution images with three-dimensional reconstruction showed the general presence of microspikes. Further, pseudopodia (proteopodia) were observed that were induced by stromal-derived factor-1 and steel factor. Proteopodia were directed towards and were quenched by stromal cells, at times bridged HSCs, and could rapidly retract or detach from cells. Proteopodia were also observed in vivo with homed HSCs in frozen sections of murine spleen, lung and heart. This is the first demonstration that HSCs are both fast and highly malleable in phenotype.
Subject(s)
Cell Surface Extensions/ultrastructure , Hematopoietic Stem Cells/physiology , Animals , Bone Marrow Cells , Cell Adhesion , Cell Communication , Cell Movement , Cell Separation , Cell Surface Extensions/drug effects , Cells, Cultured , Chemokine CXCL12 , Chemokines, CXC/pharmacology , Coculture Techniques , Female , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/ultrastructure , Image Processing, Computer-Assisted , Mice , Mice, Inbred BALB C , Microscopy, Electron , Microscopy, Fluorescence , Microscopy, Video , Stem Cell Factor/pharmacologyABSTRACT
The act of defining neuropoietic progenitor/stem cells is still in its early phases. Epidermal growth factor (EGF) stimulates extended proliferation of aggregates of subventricular striatal cells, taken from E15 mouse striatum, termed neurospheres in liquid culture. We have shown here and in previous work, using either immunohistochemistry or RT-PCR, that neurosphere cells express 13 cytokines (32 tested) and 20 cytokine receptors (28 tested), with 11 potential paracrine and nine potential autocrine loops. The neurotrophin receptors, Trk A, B, and C, were all expressed. Using a newly developed FACS single cell deposition technique, we evaluated the capacity of single EGF stimulated neurosphere cells to respond to the ligands for Trk A and B, nerve growth factor (NGF), and brain-derived neurotrophin factor (BDNF). Addition of NGF or BDNF to EGF for 14 days had no effect, but removal of EGF at day 14 with subsequent addition of BDNF or NGF resulted in an increase in neuronal and astroglial, but not oligodendrocyte, colony cells at 21 and 28 days of culture for BDNF, and of both cell types at 28 days for NGF. Tri-lineage colonies increased at day 21 with BDNF and at day 28 for both NGF and BDNF. Gross colony morphology also showed changes with neurotrophin addition, forming multiple individual cell balls or filamentous spreads. When EGF was withdrawn, a threshold effect was observed, with small, but not large, colonies ceasing growth. BDNF and NGF showed no effects on cell proliferation when compared to EGF controls, as determined by 5'-bromo-2-deoxyuridine (BrdU) incorporation and thus, they appear to affect differentiation of progenitor cells. These data indicate a sequential action of cytokines with EGF maintaining viability and proliferation and blocking differentiation. Removal of EGF is then permissive for the differentiating effects of BDNF and NGF. These data further indicate that the majority of EGF neurosphere clones have neurotrophin dependent tri-lineage potential.
Subject(s)
Cell Differentiation/physiology , Central Nervous System/embryology , Nerve Growth Factors/metabolism , Neurons/metabolism , Stem Cells/metabolism , Animals , Brain-Derived Neurotrophic Factor/metabolism , Brain-Derived Neurotrophic Factor/pharmacology , Bromodeoxyuridine/pharmacology , Cell Culture Techniques/methods , Cell Differentiation/drug effects , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured/cytology , Cells, Cultured/drug effects , Cells, Cultured/metabolism , Central Nervous System/cytology , Central Nervous System/metabolism , Cytokines/genetics , Cytokines/metabolism , Epidermal Growth Factor/metabolism , Epidermal Growth Factor/pharmacology , Fetus , Immunohistochemistry , Mice , Mice, Inbred BALB C , Nerve Growth Factor/metabolism , Nerve Growth Factor/pharmacology , Neurons/cytology , Neurons/drug effects , RNA, Messenger/metabolism , Receptors, Cytokine/genetics , Receptors, Cytokine/metabolism , Stem Cells/cytology , Stem Cells/drug effectsABSTRACT
We designed a rapid, simple and accurate PCR method to determine sexual identity of mouse fetuses collected on embryonic day 15. A multiplex PCR amplification was used to detect male-specific sequence (Sry) in DNA extracted from fetal livers through SDS denaturation followed by high salt extraction and precipitation. This extraction method resulted in sufficiently purified DNA in < 1 h and was suitable for PCR. The DNA obtained was amplified using a robot thermal cycler for 33 cycles. The reaction was performed in 50 microl, using two sets of primers specific for Sry gene (chromosome Y) and IL3 gene (chromosome 11). Amplification duration was 1.5 h. The assessment of the results was done by electrophoresis in 3% agarose run at high voltage. The 402 bp band (Sry) obtained identifies the male fetuses and the 544 bp product (IL3) confirms the correct amplification of the template DNA. The entire procedure took < 4 h. The specificity of the method was confirmed by fluorescent in situ hybridization using a specific male probe on cultured male and female neural stem cells. This method allowed the preparation and culture of pure male and female neural stem cells from fetal tissue.
