Metallica Goes The Stem Cell Way

I had previously written a little about stem cells. While researchers still don’t yet know exactly how the four factors transform the fully differentiated fibroblast cells back into pluripotency, possible explanations are pouring in.

Pluripotency (by which the stem cell may become any tissue; muscle or nerve, for example) and “self renewal” (cells should not only differentiate, some ready stock of stem cells must be there for future need) are important determinants for stem cells.

According to Shinya Yamanaka, the steps could be somewhat like this: c-Myc first confers the open chromatin state and immortality to the skin fibroblasts. But it also induces apoptosis by acting on the p53, “the guardian of the genome”. Apoptosis or cellular senescence causes the cells to die. Klf4 inhibits p53 induced apoptosis. Again, if we added only Klf4 and c-Myc we would get tumor cells (both being oncogenes). Oct4 here acts and makes ES like cells (ES= Embryonic Stem) out of what was destined to be tumor cells. Sox2 confers pluripotency and you’ve got what you wanted.

Now, we just have to hand pick the right cells from the petridish. Scientists can do it either by looking for Fbx15 expression or the expression of nanog in the treated sample. Both Fbx15 and Nanog are targets of Oct3/4 and Sox2; but Nanog is found to be more closely associated with pluripotency, as is evidenced by adult chimera formation (chimera is a monstrous fire breathing creature like dragon of ancient mythology).

There have been some important modifications. Researchers have shown that one could still get human induced pluripotent stem cells (iPSC) without the need of the c-myc oncogene. The mode of delivery of these four factors could also be undertaken by plasmids, rather than the traditional retroviral vector approach. Retroviruses (like c-Myc) could potentially induce cancer. You may like to hear this Nature Podcast where both Yamanaka and Rudolph Jaenisch give a very good summary. As a bonus, you may also appreciate another way of creating iPSC. Replace the genome in “early embryonic cells” or zygotes (fertilized eggs) during cell division. During cell division, the nuclear membrane disappears and the factors are no longer in the nucleus. They are in the cytoplasm. Dieter Egli explains that if you replaced the genome of this zygote with another (genome) while the cell was still dividing, the new genome would adapt to the new cytosolic environment and get instructions from the factors in the cytosol. It will go ‘back in time’ and become a stem cell.

Now, a bit of refreshment. Watch this awe inspiring Metallica video called 'All nightmare long'. It portrays the Tunguska event, A-bomb, Soviet Revolution, American supremacy (?) and ‘revival of organisms’. Some key phrases are:

• “like a split worm, a part of the organism can reconstitute the whole”. Check about Planarians (flat worms, picture on the left), they not only reconstitute but also become separate individuals!
  
• “Instead of offspring, they become skin cells, nerves and muscle”- just as we described! Seems Metallica is well informed! Do see this wonderful video in YouTube.

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Okita, K., Ichisaka, T., & Yamanaka, S. (2007). Generation of germline-competent induced pluripotent stem cells Nature, 448 (7151), 313-317 DOI: 10.1038/nature05934

Developmental reprogramming after chromosome transfer into mitotic mouse zygotes, doi:10.1038/nature05879

Atomic Force Microscopy: Feels The Atoms, Sees The Bonds

When it comes to viewing things on the atomic scale, one has to be very careful and innovative. To understand how an atomic force microscope works, we should better discuss a bit about its predecessor: the scanning tunneling microscope (STM). STM was invented by Binnig and Rohrer for which they got the Nobel Prize in Physics. Binnig and colleagues later went on to develop the first Atomic Force Microscope (AFM). Both AFM and STM are types of Scanning Probe Microscopy, which employs a probe that scans the sample.

STM consists of a sharp probe tip, which scans over the specimen as the adjoining picture shows. First, the probe tip is brought near the sample manually, and then the finer adjustment of maintaining probe sample distance (height) is done by piezoelectric control. A voltage applied between the tip and the sample causes electrons to tunnel from the tip to the sample. As we have seen in Ohm’s law, the tunneling current will depend on the applied potential difference (voltage bias); and the height of tip-sample separation and the local density of states (factors determining ‘resistance’). If we know two of the three unknowns, we can calculate the other, which is actually done by the computer by data acquisition.

We can do STM in two ways. We can keep the tip position (height) fixed as it scans the specimen topography (constant height mode). Here the voltage and height are both held constant, while the tunneling current varies. In constant current mode, the tip is always at a specific height over the specimen. That is, as the tip hovers over the rugged terrain of the sample surface and comes close to a raised spot, the tunneling current will increase. The increased current will be sensed, amplified and passed to the feedback electronic circuitry which will ‘lift’ the probe-tip by applying a voltage to the piezo crystal. Hence the electronic servomechanism maintains a constant tip sample distance in constant current mode. Piezoelectric crystals translate pressure changes into electricity (and vice versa) as we see in oven gas lighters and in mobile phone speakers.

But STM has its inherent drawback: the sample has to be a conductor or a semiconductor, in order for tunneling to occur. Hence, biological tissues, non conducting polymers can not be imaged. So, the need for atomic force microscopy arose. Here again, Gerd Binnig played a pivotal role.

Atomic Force Microscopy operates on a similar principle. First, let’s discuss how the music on gramophone record grooves is translated. The stylus (which overlies a piezo crystal) feels the groovy surface of the vinyl LP disc which revolves on a turntable. The mechanical vibration sets in a voltage in the piezo via the stylus. We get the surface topology in the form of music. Another great way of viewing how the AFM scans and interprets a sample is how a blind person ‘feels’ a surface by using a stick (see picture).

In AFM, a very sharp tip (made of silicon or silicon nitride) is scanned over a surface with similar feedback mechanisms that maintain the tip at a constant force (to get height information), or height (to obtain force information) above the sample surface. This sharp tip is mounted on a cantilever, a rod like structure whose other end is fixed and unmovable. As the probe tip raster scans (i.e. scans in a zigzag fashion as done in TV scanning) the surface of the sample, a laser light is made to fall on the back of the cantilever. The light gets reflected off from this side and is detected by one of a dual photodiode. The cantilever ‘tilts’ as it slopes ‘down a bump’, causing another reflection that hits the other of the dual photodiode. A differential amplifier then calculates the difference output between the two light intensities and this is proportional to the cantilever deflection. We can calculate the force (acting between the cantilever tip and the sample) using Hooke’s law (F=-kx), where F is force, k is constant for a particular cantilever, and x is the deflection of the cantilever. The amplifier difference output may be used to keep the tip in either at a constant force or at a constant height above the sample, through piezoelectric servo mechanism (feedback).

AFM is very versatile and allows many user specific modifications. It can be done in contact mode, where the tip remains in contact with the surface as it scans over the specimen. In tapping mode, the cantilever oscillates over the sample, touching the surface intermittently. Interatomic forces like van der Waals forces and electrostatic forces cause a deflection of the cantilever when the tip comes closer to the sample. In the non contact mode, the cantilever does not touch the sample, and is oscillated at slightly above its natural resonance frequency. Any long range force like van der Waals force will decrease the frequency of the vibrating cantilever, when the tip approaches the sample. There are many other variants of AFM and combination of AFM with other imaging modalities like optical microscopy, Raman spectroscopy and so on.

But Leo Gross and colleagues at IBM Research Zurich wanted another, so they went on to develop another variant of AFM. They used a tuning fork like probe. One end of the probe was near, while the other end was away from the sample. When the fork was vibrated, the prong next to the sample experienced a minute shift in frequency due to forces acting upon it. This frequency drift when compared to the other prong, gives the molecular picture.

To get the finer detail of a molecule, one had to use a sharp AFM probe tip. They linked a molecule of carbon monoxide to the tip in such a way so that the lone oxygen atom became the de facto tip. It gave the structure of pentacene in unprecedented detail. You can clearly see even the bonds between carbon atoms, just as we read in our textbooks. However, the experiment must be vibration free, thermal noise free (at a very low temperature), and should be done in vacuum.

References: Scanning tunneling microscope (Wikipedia), Atomic Force Microscope (Wikipedia),
Single molecule's stunning image (BBC)

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