Although I already commented on the stem cell discovery that came out of McMaster earlier this week, I felt that a more detailed look at the methods section would be needed to do justice to the science. After all, the true value of this discovery is in the protocol utilized to make it.
On Fibroblasts and Blood: Just to recap, Dr. Mick Bhatia and his colleagues at McMaster University published findings in Nature earlier this week explaining how they have managed to convert human skin cells to various cellular components of blood. In order to do this they first cultured human fibroblasts in a regular mix of cell culture media atop a thin layer of extracellular matrix protein known as matrigel. By supplementing the media with two growth factors essential for early hematopoiesis, FLT3LG (FMS-like tyrosine kinase 3 Ligand) and SCF (stem cell factor), and transfecting the cells with a lentivirus carrying the stem cell gene OCT4, they were able to stimulate formation of a multipotent hematopoietic progenitor expressing the lineage marker CD45+. This cell type could then be coerced into different blood cells with the addition of a few more hematopoietic cytokines; after which Dr. Bhatia observed the formation of three distinct cell types – monocytes, granulocytes, and myeloid cells, all expressing unique lineage markers. Amazingly, the monocytes could be grown in the presence of M-CSF (macrophage-colony stimulating factor) and IL-4 (interleukin-4) to produce macrophages that actually engulfed FITC-labelled beads. To produce red blood cells, EPO (erythropoietin) had to be added during the initial step of the protocol along with FLT3LG and SCF, upon which enucleated red blood cell-like cells emerged expressing the erythroblast marker CD71. The next step is figuring out how a single efficient differentiation protocol can produce the full spectrum of blood components in one shot, which will be a challenge, but one I’m sure the team at McMaster’s Stem Cell and Cancer Research Institute are up to.
Bacterial Immune System: Some bacteria have their own micro-immune system in the form of the CRISPR/Cas locus. After bacteria are infected with viruses this immune locus takes up small pieces of viral DNA known as ‘spacer’ DNA. These provide a mode of protection by allowing the bacteria to recognize and destroy foreign viral DNA upon subsequent infections. In a recent Nature study, researchers discovered that bacteria are also able to incorporate spacer DNA from plasmids that contain antibiotic resistance genes. Bacteria that do so inadvertently lose antibiotic resistance by destroying the plasmid, and as a result, are unable to pass these genes on to other bacteria. Exploitation of the CRISPR/Cas locus could allow for the generation of safer bacterial strains with greater resistance to bacteriophages and less antibiotic resistance. This study was led by Dr. Sylvain Moineau of the Department of Biochemistry at Laval University.
A Hidden Hotspot: Scientists at the University of British Columbia have recently solved the crystal structure of the ryanodine receptor found within the endoplasmic and sarcoplasmic reticulum – cellular organelles that surround the nucleus. Mutations in the receptor, which governs the release of calcium ions in muscle cells, have led to serious cardiac and skeletal diseases in humans. Several mutation ‘hotspots’ were identified in the hidden cytoplasmic domain of the receptor, explaining why scientists were previously unable to find any. The findings of Dr. Filip Van Petegem and his team, published in Nature, will allow for the development of new methods to target abnormalities in ryanodine receptors.
Robotic Precision: A new feat from the Department of Mechanical and Industrial Engineering at the University of Toronto – single cell manipulation and patterning with a robotic system. Dr. Yu Sun and his team developed motion control algorithms and integrated this with computer ‘vision’ to allow a robot to track cells in real time, pick single cells up, and then drop them off at precise locations. The machine uses a glass pipette, similar to those used in manual manipulation of cells, and can carry out its daily job at a rate of 15 seconds per cell with a 95% success rate. The device is expected to greatly bolster the speed of single-cell studies, and should prove useful for any studies requiring fine manipulation. Find the study in PloS ONE.