Twenty years ago Mary Schweitzer found herself the closest that anyone has ever been to a living dinosaur. As she examined a thin slice of a T. Rex bone fragment under a microscope, she realized she was looking at what appeared to be preserved red blood cells- cells which had no place in a 65 million year old fossil. It was the first time that anyone had found evidence that biological material could survive the passage of millions of years and still retain its molecular structure, challenging one of the central beliefs of paleontologists. 
Proving that what she was seeing had in fact once been dinosaur cells was a tall order, however, and one so outrageous at the time that it was met with considerable hostility from her peers. When Schweitzer first claimed to have found red blood cells in a T. Rex leg bone in 1993, her findings were dismissed by most scientists because it seemed improbable- not to mention downright impossible- that red blood cells could have survived in a 65 million year old bone. Schweitzer recieved her Ph.D. for her work on proving that the cells had originated from once-living dinosaur cells. The controversy was renewed in 2005 when Schweitzer reported finding collagen, a tough, elastic structural protein, in fossilized bones. Since then Schweitzer has found evidence of blood vessels, feather fibers, and osteocytes (specialized bone cells) associated with fossilized dinosaur bones. 

Many of her fellow paleontologists remain unconvinced, however, and so for the past several years the burden of proof has been on Schweitzer on her team to prove that they are dealing with true dinosaur cells. Skeptics argue that Schweitzer’s findings are really biofilms- thin, slimy films of bacteria- which adhered to the hollows and spaces within the bone that were originally occupied by cells and blood vessels. Upon dissolution of the mineral substance of the bone, they contend, the biofilms retain their original shape and simply appear to be cells or vessels. Schweitzer is battling these claims by steadily accruing proof that the cells in question are truly dinosaur osteocytes, and her most recent findings are set to appear in the journal Bone (an advanced version of the accepted manuscript is already available online, see citation below). 

In her most recent publication Schweitzer applies a number of molecular methods to demonstrate the likelihood of the cells' 'dinosaurian' origins. Schweitzer and her colleagues analyzed a Tyrannosaurus Rex bone recovered from the Hell Creek Formation in eastern Montana and a Brachylophosaurus hindlimb recovered from Judith River Sediments in northern Montana. Fragments from the bones were demineralized to remove the hardest parts of the bone, freeing the soft structural protein matrix, tissue vessels, and osteocytes trapped within the bone. These bits were what the authors were the most interested in- bone is mostly calcium and other hard minerals, and so it is pretty resistant to even millions of years of geological time, but the researchers wanted the good stuff. Soft tissue and the cellular parts of the body are usually the first to go after an organism dies, as they are so delicate that they can’t survive harsh and fluctuating environmental conditions, especially not for the amount of time that has passed between now and the time when dinosaurs roamed. They repeated the same demineralization process described above with ostrich and alligator bones taken from animals which had died several months prior (why the ostrich and the alligator, you ask? We’ll get to that later).

By using microscopes to image the cells the team found that the cells extracted from the dinosaur bones resembled osteocytes in both shape and in their location within the bone. Next, researchers started to look at the stuff inside the cell- namely, the proteins within the cell and where they were located. They stained the samples with antibodies, small proteins which ‘stick’ to specific proteins. Antibodies are actually the soldiers of our immune system, as they recognize specific proteins and molecules on the surface of foreign objects and subsequently target the invader for attack. Since their discovery, however, molecular biologists have hijacked antibodies for their own purposes. 

First, the team used antibodies against actin and tubulin to look at the distribution of each protein, as both are filamentous structural proteins which are fairly abundant in all animal cells. The distribution of actin and tubulin was similar in all four animal cells, which they had anticipated- this also proved that the putative dinosaur osteocytes could not be biofilms, as bacteria do not have the same structural proteins. To reinforce this they stained bacteria which had been isolated from the soil surrounding the samples but there was no antibody binding, indicating that there was no detectable actin or tubulin. 

In a clever bit of molecular and evolutionary ingenuity, the researchers set out to prove that the cells were most likely dinosaur osteocytes- a tall order when there is no other known preserved dinosaur cells, and they couldn’t very well go out and take a sample from a passing dinosaur. Dinosaurs are most closely related to modern day birds (of all things), and so the authors speculated that if they were dealing with true dinosaur osteocytes then the cells would share some molecular properties with avian osteocytes. Thus, they decided to include the ostrich in their study as a point of reference and a positive control. 

The researchers then selected an antibody which detects a protein (PHEX) that is slightly conserved- meaning that is has regions which are similar in structure- throughout all vertebrate species, however, the particular antibody they used only detects the part of the PHEX protein which is unique to avian osteocytes. They reasoned that the dinosaurs may be related enough that the avian antibody would also be able to detect the dinosaur antibody, as is sometimes the case among evolutionarily proximal species. 

Indeed, cells from both dinosaurs had a similar PHEX staining pattern as the ostrich osteocytes: the antibody detected PHEX protein around the surface of the cell, and on the filopodia, which are thin, sheetlike surface projections. The alligator had been selected as a negative control, to prove the specificity of the antibody to avian osteocytes. As expected, the alligator sample had no detectable antibody binding. The fact that the antibody bound to a protein present on the cells taken from both sets of dinosaur bones implies that the cells in question most likely originated from dinosaurs. 

Next, they demonstrated the presence of DNA within both sets of dinosaur osteocytes by staining the samples with a chemical that jams itself in between DNA base pairs and glows when fluorescent light is shone on it. Not only was there DNA- albeit in relatively low levels, as most had probably been degraded over time- the authors also confirmed the presence of a DNA-associated protein in these cells which is specific to higher-order animal cells, another strike against the bacterial biofilm hypothesis. The limited amount of DNA in the cell makes DNA sequencing difficult, but the authors suggest that the DNA binding proteins could be taken from the cells and that the bits of DNA stuck to them could be sequenced.

The big question, then, is how these fragile cells managed to survive in an environment where everything has been working against them for 65 million years. The authors are still at a loss to explain such a remarkable feat, although they speculate that the fact that these cells are entombed in bone is most likely key to their survival. Embedded deep within the bone, the osteocytes are protected from other live cells and microbes; osteocytes in particular are not dividing cells and so they are expected to live roughly the lifetime of the organism (of course, this is not always the case), therefore they are more resistant to programmed cell death; and the fact that they are esconced in mineral may serve as a protective feature. Interestingly, the researchers also hypothesize that the iron released from red blood cells after they die may have ‘fixed’ the cells and their proteins in place by forming an elaborate crosslinking bridge between iron molecules, cell membranes, and associated proteins, keeping everything firmly secured in place and resistant to degradation.

In addition to the Bone paper which was recently accepted for publication, Schweitzer also gave a talk at the annual meeting of the Society for Vertebrate Paleontology on October 17th. It is not yet clear how skeptics will react, although many of her findings are certainly damning to the biofilm hypothesis. Most scientists like to exercise exceptional prudence, however, especially when dealing with something as improbable as dinosaur cells surviving for millions of years, and so Schweitzer may still have some work to do before the 'dinosaurian' origin of the cells becomes widely accepted. 

Schweitzer Mary Higby, Zheng Wenxia, Cleland TimothyP., Bern Marshall, Molecular analyses of dinosaur osteocytes supports the presence ofendogenous molecules, Bone (2012), doi: 10.1016/j.bone.2012.10.010