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Sunday, October 17, 2010

Sensory Renewal: Goldfish Eyes & Hair Cells

The U.S. Census Bureau reports in its results from the 2006 American Community Survey that 7 million Americans older than five years possess a severe sensory disability. According to the National Eye Institute, 1.75 million Americans are losing vision, owing to age-related macular degeneration. In the year 2020, their ranks will swell to roughly 3 million. Gallaudet University suggests in informative, well-documented data analyses that about 1 million Americans are functionally deaf. Recent breakthroughs in regenerative medicine have decisively progressed toward promising treatments.

Carassius auratus (courtesy G. Blakemore)
I once was involved in research on plasticity of the goldfish retina (a micrograph of a beautifully stained cross section through a chicken retina similar to fish is shown here). Carassius auratus grows in size throughout its lifetime. Many of us may have encountered well-fed goldfish as long as a lower arm. With eyes growing continuously, the retina must keep up. It does not merely stretch. Rather, dividing pluripotent precursor stem cells are retained in a marginal zone, from which the needed retinal cells differentiate.

Retinal ganglion cells constitute the nerve cells that convey visual information from the retina to the optic tectum in the midbrain where their endings terminate in a topographic map of the retina. Because of this retinotopy, an image of the surrounding world cast on the retina retains its spatial relations when it is represented by nerve cell activity in the tectum. As the retina grows, retinotectal nerve fibers are added and new tectal terminations are formed, while old ones continue to process visual information. In our mature visual system nothing like this happens. Once our brain matured, the retinal nerve cells and their connections with the brain remain by and large unchanged.

In addition, the goldfish visual system is capable of an achievement even greater than the continuous addition of functionality. Maier and Wolburg (1979) discovered that goldfish retinae deprived of almost all cells by metabolic poisoning were able to reconstitute themselves from scratch within a few months. As we know now, surviving Müller glia cells and photoreceptor cells convert into multipotent stem cells that serve as dividing progenitors from whose offspring retinal cells differentiate  (Bernardos and others, 2007). The new ganglion cells innervate the optic rectum in roughly topographic fashion. My project was to examine with a functional imaging method whether the newly formed connections with the tectum were functional. Indeed, my colleagues and I found compelling evidence that the novel retinotectal inputs could be activated by visual stimulation.

Destruction & Renewal (Melzer & Powers, 2001)
The figure above shows pseudo-colored nerve cell activation (blue - low; red - high) in slices cut transversely through the fish brain at the level of the optic tectum, that is the outer rim transected by black lines on both sides (the bar in the lower left pertains to 0.5 mm; the fish's top is up; the fish's right side is on the left). The right eye had been injected with ouabain, a metabolic toxin that kills retinal nerve cells. The retinotectal pathway is crossed. Stimulation with black and white stripes of all orientations resulted in strong activation of the optic tectum on the side receiving input from the intact left eye (red band in the tectum on the left). One week after the ouabain injection (1w p.o.), the nerve cell response on the side that used to receive input from the poisoned eye was distinctly reduced (yellow band on the right). Fourteen weeks after ouabain injection (14w p.o.), tectal activation had recovered noticeably, owing to the reconstitution of the poisoned retina and the regrowth of retinotectal connections (Melzer and Powers, 2001).

Alas, the terminally differentiated cells in our mature retina do not possess the goldfish's power of spontaneous regeneration. However, much progress has been made since I conducted my research, unraveling the molecular mechanisms and identifying the genes that reconvert differentiated cells into multipotent stem cells or conditioned pluripotent embryonic stem cells to become progenitors. Recently, Advanced Cell Technology developed the first gene therapy for people with Stargard's macular dystrophy that will soon be tested in clinical trials, aiming to replace dysfunctional pigment epithelium cells in the retina with healthy ones generated from human stem cells.

By contrast, the effort to restore hearing in deaf people has not advanced to clinical trials yet, though stem cells have also shown promise. Oshima and others (2010) successfully directed induced pluripotent stem cells and embryonic stem cells from mice to change into skin cells. The skin cells were subsequently converted into progenitor cells. The researchers were then able to promote these progenitors' offspring to differentiate into hair cell-like cells with stereocilial bundles that exhibited stimulus transduction currents resembling those of immature sensory hair cells in the organ of Corti of the cochlea, that is the inner ear.
Schematic crossection through the organ of Corti showing the hair cells topped by the basilar membrane surrounded by support cells (courtesy Madhero88).
The organ of Corti is situated roughly in the middle of the cochlea running in its turns from base to apex. Sound vibrations are conveyed from the tympanic membrane via three small bones in the middle ear to the cochlea. The cochlea is filled with liquid. The sound vibrations move the organ of corti's tectorial membrane atop the hair cells, deflecting the cilia. The hair cells transduce the deflections into tiny electrochemical currents and pass them onto nerve cell endings of the auditory nerve the fibers of which convey the resulting electrical impulses to the brain. With decreasing sound frequency, the location on the organ of Corti that is stimulated most strongly shifts from the cochlea's base to its apex, providing the mechanical foundation for our ability to discriminate pitch.  The representation of pitch in discrete regions ordered by frequency is called tonotopy and remains preserved in all information-processing stations of the brain's central auditory pathway (I have written about tonotopy in my previous posts dated Sep. 30, 2009, and Oct. 12, 2009).

Only about 14,000 hair cells populate the human organ of Corti. Because of the inability of our hair cells in the inner ear to repair or replace themselves, drug- or sound volume-induced damage irreversibly leads to the persistent loss of hearing in the range of pitch associated with the location of the damage on the organ of Corti. In offering one possible resolution of this predicament, Oshima and others (2010) recently developed a three-step hair cell generation procedure, manipulating molecular signaling pathways that regulate cell fate and proliferation in tissue culture. In the first step, inhibition of the Wnt/TGF-beta pathways commits the stem cells to ectoderm. In the second step, FGF signaling promotes the ectodermal cells into otic progenitors. In the third step, the progenitors divide and differentiate into immature hair cell-like cells in a culture system with stromal cell-derived activity. The findings constitute a proof of concept, demonstrating that the replacement of hair cells from stem cells is possible. Whether the newly generated cells can be implanted in inner ears and form functional connections with nerve cells remains to be established.

Alternatives to the implantation of stem cells may exist. In vertebrates other than mammals lost hair cells are replaced by regeneration from support cells in the surrounding skin, known as phalangeal cells and Deiters' cells. The support cells convert into dividing pluripotent otic progenitor cells. In analogy, methods that may help de- or transdifferentiate support cells into dividing progenitor cells in vivo are being pursued (Izumikawa and others, 2005; Löwenheim and others, 1999). 

Regardless of the obstacles that still need to be overcome, there is now a good chance that stem cell therapies or regenerative medical procedures derived from this research may be able to help people with sensory disabilities regain function.

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Addenda
  • The picture of the retina mentioned above and other astounding renderings of the brain can found in Carl Schoonover's recent book with the title "Portraits of the Mind: Visualizing the Brain from Antiquity to the 21st Century" (11/07/10).
  • Today the Swedish Royal Academy awarded this year's Nobel Prize in Physiology or Medicine to John B. Gurdon from Great Britain and Shinya Yamanaka from Japan for their successes in converting differentiated cells into pluripotent stem cells (10/08/2012).
  • Almost a decade ago, Izumikawa and others (2005) showed in guinea pigs that the administration of adenoviral vectors containing the gene that encodes ATOH1, a transcription factor crucial for hair differentiation, to non-sensory support cells in the organ of Corti resulted in the regeneration of hair cells and recovery of hearing. Helen Thomson reports in her post with the title “Deaf people get gene tweak to restore natural hearing” published online by the New Scientist Apr. 23, 2014, that the first clinical trials using the principle of this method are going forward at the University of Kansas Medical Center (04/25/2013).
  • Schwartz and others (2012) have been testing ACT’s therapy. NPR’s Rob Stein reported in his Morning Edition segment published Oct. 14, 2014, with the title “Embryonic Stem Cells Restore Vision In Preliminary Human Test” for Morning Edition improvement of eyesight in 10 of 18 patients enrolled in the phase I trial (10/15/2014).

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