Hair Cell Regeneration: 15 Years Later and What do We Know?

1. Hair Cell Regeneration: 15 Years Later and What do We Know? Brenda M. Ryals, Ph.D. James Madison University West Virginia Speech Language Hearing Association March 30, 2006

2. The discovery of hair cell regeneration in 1988 contradicted everything we knew about hair cell loss and permanent sensorineural hearing loss.

3. Hair cell regeneration:  An exciting phenomenon, but will it be possible to restore hearing and balance? Matsui and Ryals (2005) Jul-Aug;42(4Suppl 2):187-98 A Primer on Biology of Hair Cell Regeneration, Rescue, and Repair, Ryals and Cunningham (2003) 24(2):99-110

4. How was Hair Cell Regeneration confirmed in Birds? Using markers for DNA replication investigators labeled cells which were newly formed after trauma (Corwin & Cotanche 1988;Ryals and Rubel 1988) Tritiated thymidine marker BrDU marker

5. Where did these new hair cells come from? A population of progenitor cells exist in birds which are capable of re-entering the cell cycle to divide asymetrically and differentiate into hair cells Tritiated thymidine marker BrDU marker

6. Birds What is involved in “making a new hair cell”? Regenerating Repaired Normal TIME

7. Birds 1) Renewed cell division 2) Cell fate determination 3) Cellular maturation

8. Direct cell conversion: SC HC

9. So here’s what we know about hair cell regeneration in birds: • Precurser cells (supporting cells) are triggered to re-enter the cell cycle when hair cells are damaged or destroyed • These newly produced cells are signaled to differentiate into hair cells • Finally the new hair cells mature and are innervated

10. Factors involved in regulating Cell Cycle

11. Factors involved in regulating Cell Cycle • Genetically controlled tumor suppressor proteins can inhibit or stimulate cell cycle and Growth factors generally stimulate re-entry into cell cycle.

12. Factors involved in regulating Cell Cycle • One way that growth factors might enter the inner ear after damage or hair cell loss is through macrophage activity. • Macrophages are specialized white blood cells that actively secrete growth factors to stimulate wound repair.

13. Factors involved in regulating Cell Cycle • So in birds we have precursor cells which respond to either to a release of inhibition (genetically controlled tumor suppressor) AND/OR • Respond to the stimulatory effects of growth factors

14. Audiologists Want to Know: • Why can’t mammals/humans automatically generate new cells after hair cell damage or loss? • A. There are no endogenous precursor cells in mammalian cochlea • B. Growth factors can’t enter the cochlea because of “blood brain barrier” • C. Tumor suppressor genes strongly inhibit re-entry into the cell cycle • D. Cell fate determinants (morphogens) aren’t available to guide hair cell differentiation

15. Break-out Questions • Why can’t mammals/humans automatically generate new cells after hair cell damage or loss? • X. There are no endogenous precursor cells in mammalian cochlea

16. Mammalian support cells and pillar cells CAN be induced to divide and become hair cells. • They ONLY do this “naturally” given two circumstances: • Release of genetic inhibition • Presence of genetic signal for hair cell differentiation Doetzlhofer et al. Dev. Bio. 272 (2004) 432–447; Doetzlhofer et al ARO 2005

17. Audiologists Want to Know • Why can’t mammals/humans automatically generate new cells after hair cell damage or loss? • X. Growth factors can’t enter the cochlea because of “blood brain barrier”

18. Macrophage activity in the cochlea after injury (Warchol 1997, Bhave et al 1998) providing growth factors which may trigger progenitor cell division or phenotypic conversion Cell death in the cochlea (apoptosis) may release factors which stimulate cell division - evidence indicates a reasonably linear relationship between cell death and cell division/conversion

19. Audiologists Want to Know • Why can’t mammals/humans automatically generate new cells after hair cell damage or loss? • C. Tumor suppressor genes strongly inhibit re-entry into the cell cycle

20. P27 kip1 (tumor suppressing protein) associated with hair cell generation: Mice deficient in the gene that regulates this protein developed too many hair cells (Segil et al 1999)

21. Audiologists Want to Know • Why can’t mammals/humans automatically generate new cells after hair cell damage or loss? • C. Cell fate determinants (morphogens) aren’t available to guide hair cell differentiation

22. Factors involved in regulating Cell Cycle - promotion of cell differentiation or cell fate • Math1 (Bermingham et al 1999) more recently termed Atoh1 • Hes1 (Zheng et al 2000) Genes available embryonically but “turned off” in mature animals

23. Retinoblastoma gene (pRb1) a tumor suppressing protein related to p27kip1 has also been associated with hair cell generation in mammals: Mature hair cells in mice with a targeted deletion of the retinoblastoma protein were able to re-enter cell cycle, divide and produce new hair cells (Sage et al 2005)

24. So here’s what we know about why mammals don’t normally regenerate hair cells after injury or death: • Precurser cells are present but under strict inhibitory genetic control so that they do not re-enter cell cycle • Morphogens which normally signal cell fate are “turned off” in the mature cochlea

25. Audiologists Want to Know • Have there been any studies which have been successful in stimulating hair cell regeneration in mammals? A. Studies using agents to remove the inhibitory influences of tumor suppressor genes or stimulate the excitatory influences of growth factors B. Studies using stem cells C. Studies using gene therapy

26. Audiologists Want to know Have there been any studies which have been successful in stimulating hair cell regeneration in mammals? • Studies using agents to remove the inhibitory influences of tumor suppressor genes or stimulate the excitatory influences of growth factors

27. Audiologists Want to Know Have there been any studies which have been successful in stimulating hair cell regeneration in mammals? D. Studies using Stem Cells

28. Stem Cells Definition: Stem cells are characterized by their capacity to self-renew and their ability to differentiate asymmetrically to form cell types other than their own.

29. From Parker and Cotanche 2004

30. Stem Cells and the Cochlea Li et al., Nature Medicine 9:1293, 2003 Heller, Li and colleagues have isolated stem cells from the mammal vestibular epithelium and shown they make hair cells when transplanted into the chick otocyst. They have also coaxed mouse embryonic stem cells down a hair cell pathway and shown that they also make new hair cells when transplanted into the chick otocyst. Li et al., PNAS 100:13495, 2003

31. Limitations of stem cell use? • Availability • Ectopic hair cells • Integration into site of lesion • Innervation

32. Audiologists Want to Know Have there been any studies which have been successful in stimulating hair cell regeneration in mammals? D. Studies using gene therapy

33. What is Gene Therapy? • Gene therapy is an experimental treatment that involves introducing genetic material into a person’s cells to fight disease • A gene can be delivered to a cell using a carrier known as a “vector.” The most common types of vectors used in gene therapy are viruses.

34. Websites with explanations of Gene Therapy • Human Genome Project(http://www.ornl.gov) • National Cancer Institute • (http://www.cancer.gov/)

35. What kind of gene would help hair to restore hair cells to a damaged cochlea? • Math1* (Bermingham et al 1999) *now known as Atoh1 • Hes1 (Zheng et al 2000) • Delta and Notch signaling (Stone and Rubel 1999) • Retinoic Acid (Kelly et al 1995

36. Gene Therapy in the Cochlea Yehoash Raphael and colleagues (2003) injected gene for Math1 into damaged guinea pig cochleae and saw new hair cells develop in damaged regions. In their second experiment they confirmed functionality of hair cells with ABR (2005) Kawamoto et al., J. Neurosci 23:4395, 2003 Izumikawa et al Nat.Med. 2005

37. Experimental design Scalamedia Infusion pump Adult guinea pigs deafened with kanamycin/ ethacrynic acid 5 ml Ad.vector 2nd turn of cochlea • Ad.Math1 or Ad.Math1-GFP • Ad.empty or Ad.GFP Experimental groups

38. Limitations of Gene Therapy in the Inner Ear • Depletion of important endogenous cell types (supporting cells, pillar cells, etc) • Electrical environment of hair cells/stria vascularis • Immune response • Problems with viral vectors • Innervation

39. We’ve come a long way toward reaching our GOALTo repair the damaged cochlea either by stimulation of endogenous cells to regenerate damaged tissue (cell cycle controls) or by the injection of exogenous agents, such as genetically engineered viral vectors, progenitor or stem cells to replace damaged tissue.

40. Do new hair cells restore hearing? What about speech? Brenda M. Ryals, Ph.D. James Madison University West Virginia Speech Language Hearing Association March 30, 2006

41. Hearing Loss in AdulthoodBehavioral Questions • Does the world sound the same with new hair cells? • In other words, does a “new” auditory periphery result in sufficient functional recovery that the animal can perceive and learn new complex acoustic communication signals?

42. Hearing Loss in AdulthoodBehavioral Questions • Does the world sound the same with new hair cells? • Behavioral measures include: • absolute threshold sensitivity • relative threshold sensitivity (difference limens for intensity and frequency) • perception of complex vocalizations (speech recognition)

43. Hearing Loss in AdulthoodBehavioral Questions • Can new hair cells support normal vocal behavior? • In other words, does the “new” auditory periphery result in sufficient functional recovery that the animal can learn and produce “correct” acoustic communication signals?

44. Hearing Loss in AdulthoodBehavioral Questions • Can new hair cells support normal vocal behavior? • Behavioral measures include: • complex call production template matching • Dooling et al 1997

45. Changes in Auditory Perception after Hair Cell Regeneration Behavioral testing – Set up

47. Normal Minimal Audibility Curve

48. Hearing Loss in AdulthoodBehavioral Questions • Does the world sound the same with new hair cells? 1. Changes in absolute sensitivity

49. Effect of Kanamycin (200mg/kg/day) on Hair Cells