Re"evolutionary" regenerative medicine.

THE POTENTIAL TO REGENERATE DAMAGED LIMBS AND hearts seems the subject of science fiction, but newts and zebrafish do it all the time. What can scientists learn from these simple creatures? Why have mammals not retained this remarkably useful property in the course of evolution? Can an evolutionary perspective on the mechanisms used by “lowly” organisms inform the approach to human tissue regeneration? Could this lead to the generation of abundant patient-specific differentiated cells for cell therapy, for elucidating disease mechanisms, for therapeutic drug screening? Recent studies suggest that this is possible. The newt’s ability to regenerate its heart or an entire limb after amputation has attracted the attention of scientists for centuries, with the hope that it might be possible to mimic this remarkably useful capacity in humans. Pioneering work with this species has provided many seminal insights. Limb regeneration entails regrowth of a diversity of tissues in their appropriate positions and proportions. This complex process is dependent on innervation and requires the orchestration of a dynamic interplay of cells. Following amputation of a newt limb, a blastema, or cell aggregate, forms from which the limb develops. The blastema was long thought to comprise a cluster of multipotent cells that had to specialize anew. However, recent elegant lineage tracing studies have shown that the cells are not multipotent but instead remain dedicated cartilage, bone, neural, and muscle cells. A critical step in this process of limb regeneration is the acquisition of proliferative potency. This is achieved by reentry into the cell cycle of postmitotic cells that retain their specified identity. A similar process is also observed in zebrafish heart regeneration. What if, as in newts, fully specialized, nondividing human cells could dedifferentiate and be pushed back just one step to a proliferative state? While retaining their identity or “sense of self,” these cells could yield precise copies of themselves capable of regenerating the damaged tissues from which they arose. A crucial step would likely entail “lifting the brakes” on cell division, but only transiently, to avoid uncontrolled proliferation and tumor formation. How could dedifferentiation be achieved? Transient inhibition of tumor suppressors might play a role. It has been postulated that during evolution mammals lost regenerative potential as a trade-off for cancer protection. The tumor suppressor Rb, encoded by the retinoblastoma gene, is known as the eukaryotic “gatekeeper” of the cell cycle G1-S transition. Inactivation of Rb mediates newt regeneration. In contrast, loss of Rb does not lead to mammalian cell dedifferentiation (eg, primary skeletal muscle cells). What might the additional mammalian brake on cell cycle reentry be? Cancer biologists had previously defined a critical function for the alternative reading frame protein (ARF; also known as p19ARF in mice and p14ARF in humans), a product of the mammalian ink4a tumor suppressor locus. ARF enforces cell cycle arrest and prevents tumorigenesis when Rb is inactivated (either by signaling or by mutation). In human cancers, ARF is frequently inactivated. Even mature differentiated cells can become transformed when ARF is inactivated, if exposed to aberrant growth factor signals. In evolution, ARF has no homologues in regenerative vertebrates (in contrast with the other product of the ink4a locus, p16). Indeed, ARF has not been identified in organisms lower on the evolutionary tree than chickens. Thus, ARF was postulated to be the culprit. Remarkably, on transfection of inhibitory RNAs to both Rb and ARF, primary muscle cell nuclei initiated DNA synthesis. Thus, the double knockdown of these 2 tumor suppressors overcame the block to cell cycle reentry. A critical remaining question was whether cells that reentered the cell cycle could complete mitosis and proliferate following treatment with inhibitory RNAs targeting Rb and ARF. This was examined by testing differentiated mononuclear muscle cells, known as myocytes, that will not reenter the cell cycle regardless of the barrage of growth factors to which they are exposed. To show definitively that a postmitotic cell could initiate division, a means of following single cells was essential. Using live cell laser capture catapulting, single myocytes were circumscribed and cut out