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

The discovery of hair cell regeneration in 1988 contradicted

everything we knew about hair cell loss and permanent sensorineural

hearing loss.

A Primer on Biology of Hair Cell Regeneration, Rescue, and Repair, Ryals and Cunningham (2003) 24(2):99-110

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

How was Hair Cell Regeneration confirmed in Birds?

Using markers for DNAreplication investigators labeled cells which were newly

formed after trauma (Corwin & Cotanche 1988;Ryals and Rubel 1988)

BrDU marker Tritiated thymidine marker

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

BrDU marker Tritiated thymidine marker

BirdsWhat is involved in “making a new hair cell”?

TIME

Direct transdifferentiation

S

M

G1G2

Mitotic regeneration

Normal Regenerating Repaired

Birds

1) Renewed cell division

2) Cell fate determination

3) Cellular maturation

Direct transdifferentiation

S

M

G1G2

Mitotic regeneration

Direct cell conversion: SC HC

Direct transdifferentiation

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

Direct transdifferentiation

Factors involved in regulating Cell Cycle

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.

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.

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

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

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

Doetzlhofer et al. Dev. Bio. 272 (2004) 432–447; Doetzlhofer et al ARO 2005

•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

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”

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

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

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)

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

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

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)

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

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

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

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

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.

From Parker and Cotanche 2004

Stem Cells and the Cochlea

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., Nature Medicine 9:1293, 2003

Li et al., PNAS 100:13495, 2003

Limitations of stem cell use?

– Availability

– Ectopic hair cells

– Integration into site of lesion

– Innervation

Audiologists Want to Know

D. Studies using gene therapy

Have there been any studies which have been successful in stimulating hair cell regeneration in mammals?

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.

Websites with explanations of Gene

Therapy– Human Genome Project (

http://www.ornl.gov)

– National Cancer Institute

– (http://www.cancer.gov/)

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

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, 2003Izumikawa et al Nat.Med. 2005

Experimental designExperimental design

1. Ad.Math1 or Ad.Math1-GFP

2. Ad.empty or Ad.GFP

Adult guinea pigs deafened with

kanamycin/ ethacrynic acid

Infusion pump

5 5 l Ad.vectorl Ad.vector

2nd turn of cochlea2nd turn of cochlea

Experimental groupsExperimental groups

Scala media

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

We’ve come a long way toward reaching our GOAL

To 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.

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

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?

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)

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?

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

Changes in Auditory Perception after Hair Cell RegenerationBehavioral testing – Set up

Normal Minimal

Audibility Curve

Frequency (kHz)

0.1 0.3 0.6 1 2 4 6 10 30

Abs

olut

e th

resh

old

(dB

SP

L)-20

0

20

40

60

80

100

Canary

Human

Cat

Hearing Loss in AdulthoodBehavioral Questions

• Does the world sound the same with new hair cells?

1. Changes in absolute sensitivity

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

Days

0 5 10 15 20 25 30

Abs

olut

e T

hres

hold

dB

SP

L (r

e 20

pa

)

0

10

20

30

40

50

60

70

80Injections

PTS of 23 dB

125 250 500 1000 2000 4000 8000Frequency in Hz

0

10

20

30

40

50

70

60

80

90

100

Heari

ng L

evel in

dB

SPL

X XX X X X = pre-injection

XX

X

XX

X = 2 weeks post injection

X XX

X

X X= 8 weeks post injection

Example audiogram after Kanamycin injections

Hearing Loss in AdulthoodBehavioral Questions

• Does the world sound the same with new hair cells? – 1. Changes in absolute

sensitivity

Conclusion: Absolute sensitivity returns to normal for lower frequencies with a some permanent threshold shift for highest frequencies

Discrimination

Effect of Kanamycin on Frequency and Intensity

Difference Thresholds

Pre 4-6 8-10 12+ Pre 4-6 8-10 12+0

2

4

6

I (

dB)

Weeks

1.0 kHz 2.86 kHz

Pre 4-6 8-10 12+ Pre 4-6 8-10 12+0

10

20

30

F (

Hz)

Weeks

1.0 kHz 2.86 kHz

A

B

Hearing Loss in AdulthoodBehavioral Questions

• Does the world sound the same with new hair cells? – 1. Changes in relative sensitivity

Conclusion: Difference limens for both intensity and frequency return to normal 4-6 weeks following hair cell loss and regeneration

Fre

quen

cy (

kHz)

Time (ms)

What is the effect of regeneration on perception and production of relevant, complex acoustical stimuli?

Effect of Kanamycin on Call Recognition

-5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 24 38 39 40 41 42 43 44

Pe

rce

nt C

orr

ect

0

40

60

80

100

Pre-Injection

Injections

Recovery

Hearing Loss in AdulthoodBehavioral Questions

• Does the world sound the same with new hair cells? – 1. Changes in perception of

complex vocalizations (speech recognition)

When the papilla first becomes repopulated, the world sounds different. But, after several months of recovery, there are no demonstrable perceptual deficits

Vocal production in budgerigars Kazu Manabe, Michael Osmanski, Ashwin Plachikkat, Manjit Sahota

Training birds to produce specific vocalizations by operant conditioning

Lombard effect

Effect of Kanamycin (200mg/kg/day) on Vocal Precision

0 5 10 15 20 25

0.6

0.7

0.8

0.9

1.0

Injections

Days

Rel

ativ

e S

imila

rity

Hearing Loss in AdulthoodBehavioral Questions

• Purpose II - Can new hair cells support normal vocal behavior?– The precision of vocal

production is only temporarily disrupted and only when hearing loss is the most severe.

Summary and Conclusions

• We know a lot now about hair cell regeneration in non-mammals and we are just beginning to see the fruits of our efforts to induce hair cell regeneration in the mammalian cochlea

• While inherent genetic inhibitory controls prohibit spontaneous hair cell regeneration in mammals, recent discoveries suggest that the use of externally introduced factors and/or stem cells may override this inhibition.

Summary and Conclusions

• Safe and effective means of delivering genes and/or stem cells need to be developed in order for these therapies to be of any clinical relevance.

• Moreover, being able to direct stem cells and/or viral vectors to the correct locations to promote supporting cell proliferation and then hair cell differentiation is paramount to the return of function.

Summary and Conclusions

• Finally, the impact of induced hair cell regeneration on neural connections and the brain will need to be understood in order to predict the ultimate impact of mammalian hair cell regeneration on restoring the complex mechanisms involved in hearing and understanding human speech.

Questions Audiologists Often Ask:

•When will hair cell regeneration be a reality for my patients?

•How will we determine candidacy?

•What will the impact of hair cell regeneration be in patients who are or have been candidates for hearing aids or other amplification devices?

•Will hearing aids or cochlear implants continue to be necessary in the face of hair cell regeneration?

We’ve come a long way in 16 years But we aren’t done yet!

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