Derivation of cochlear cells from pathological or isogenic human iPSCs for modeling hereditary hearing loss

(1)

Amandine CZAJKOWSKI

1 , Grobarczyk Benjamin

1 , Laurence Borgs

1 , Kevin Hanon

1 , Philippe Lefebvre

2 ,

Laurent Nguyen

1 and Brigitte Malgrange

1

1. GIGA-Neurosciences, Developmental Neurobiology Unit, University of Liège, Belgium

2. Department of Otorhinolaryngology, University of Liège, Belgium

Derivation of cochlear cells from pathological or isogenic human

iPSCs for modeling hereditary hearing loss.

Alström Syndrome (AS) is a human autosomal recessive genetic disorder characterized by numerous clinical symptoms

including deafness. AS is caused by mutations in the ALMS1 gene encoding for ALMS1 protein which is expressed at the basal

body and implicated in ciliogenesis, cell cycle and proliferation (Jagger et al., 2011; Zulato et al., 2011 & Shenje et al., 2014).

We are interesting in understanding the unknown mechanisms involving this protein in the genetic deafness of AS patients. To

develop a model as closer as possible to the human pathology, we are using human induced pluripotent stem cells (hiPSCs)

generated from fibroblasts of healthy and AS patients. Using a stepwise protocol, we demonstrated that healthy hiPSCs

(waiting for isogenic hiPSCs) can generate a population of cells with gene and protein expression patterns consistent with the

ones of otic progenitor cells (OSCs). In these OSCs, we observed some proliferation defects and an increase in apoptosis tin the

AS cells compared to the WT. When human OSCs are co-cultured with mouse feeder cells, they are able to differentiate into

hair cells (HCs). We successfully differentiated AS hiPSCs into HCs. We are currently confirming gene expression pattern and

testing HCs functionality. To exclude patient linked epigenetics and differentiation defects, we are correcting the genomic

mutation in the AS hiPSCs to generate isogenic hiPSCs using the CRIPSR/Cas9 system. Thanks to the isogenic hiPSCs we will be

able to confirm that these defects are well due to the ALMS1 mutation.

AS hiPSCs AS OSC (CRISPR/Cas9) isogenic WT hiPSCs isogenic WT OSCs Healthy HCs genome editing Pathological HCs AS patient

1

5

2

3

Fibroblasts from healthy (WT) and deaf AS patients (AS) were reprogrammed into human induced pluripotent stem cells (WT and AS hiPSCs). Fibroblasts were infected by Sendaï virus particles containing the Oct4, Sox2, Klf4 and C-myc mRNAs. HiPSCs were then characterized before being differentiated into cochlear cell types. Reprogrammed hiPSCs expressed stem cell markers such as NANOG, OCT4, SOX2 and TRA-1-81 by immunofluorescence (1-A, 1-B) and qRT-PCR (1-C). (1-D) 8 weeks after injection in a nude mice, hiPSCs formed teratoma containing cells from the 3 germ layers.

Hoechst NANOG OCT4 NANOG/OCT4 Merge

hiPSCs AS G1608A

hiPSCs AS 707CVD

hiPSCs AS F2 hiPSCs WT

Colonies of hiPSCs expressing stem cell markers

hiPSCs NANOG OCT4 SOX2 Healthy donor WT hiPSCs reprogramming AS patient AS hiPSCs Sendaï virus

OCT4, SOX2, KLF4 & cMYC

1-A

Induction of otic progenitor cells (OSCs)

The stepwise guidance protocol is based on the inner ear embryonic development and adapted from Oshima et al, 2010 and Chen et al, 2012. Firstly we induced differentiation of hiPSCs into anterior ectoderm by blocking WNT and TGFb pathway and adding IGF1 to the culture medium. Ectodermal cells were then pushed toward an otic fate by high doses of bFGF until day 20. Around 20% of cells observed by immunofluorescence at day 9 were positive for PAX2 and the otic markers PAX8 and SIX1 meaning that these cells were OSCs. This number increased until day 20 to reach 30% (2-B). We further characterized the obtained OSCs by immunofluorescence (2-A) and qPCR (2-C). We measured the expression of PAX6, a transcription factor specifically expressed in optic progenitors and neurectoderm cells but not in OSCs as control. In presence of SU4802, a bFGF signaling inhibitor, OSCs were not induced as shown by IF and qRT-PCR.

At day 20, we changed the medium to have proliferative conditions. This culture medium maintains OSCs in long term culture. These OSC cultures still expressed PAX2, PAX8, SOX9 and Ki67 (2-E). These results obtained by immunofluorescence were confirmed by qRT-PCR (2-D) errors bars represent

Hair cells (HCs) differentiation

We cultured WT OSCs on dissociated mouse embryonic cochlear cells (from E14 embryos) which act as feeder cells to provide a kind environment for the terminal differentiation of the human OSCs into HCs. After 10 days of co-culture, we observed by IF some clusters of double positive ATOH1/MYOSIN6 cells (2-F). Using a specific antibody against human nuclei we confirmed that these cells were human OSCs differentiated and not feeder cells. HCs were surrounded by mouse cells co-expressing SOX9 and SOX2, two markers of supporting cells in the organ of Corti (2-G). Recently, we obtained interesting results regarding the functionality of differentiated HCs using FM1-43 (2-H). This dye has the property to enter specifically into HCs by the mechanical channels so to label only functional HC.

Otic progenitors co-express PAX2, PAX8, SOX2, SIX1 and NOT PAX6

bFGF as specific otic inducer Healthy donor WT hiPSCs _{WT OSCs} otic induction βFGF, Heparan sulfate Day 20 Day 0 Day 0 Day 6 Day 9 Day 20 Pax22

PAX2+ OSCs increase from day 9 to day 20

WT OSCs

Hoechst PAX2 PAX8 Pax6 Pax2/Pax8

40x

Hoechst Ki67 SOX9 SOX1 Ki67/SOX9

40x

P4

P2 From hiPSCs to OSCs

Terminal differentiation into WT HC

HOECHST ATOH1

hNUCLEI MYOSIN6

ATOH1/MYOSIN

6 MERGE

WT OSCs P4 on mEOC – Day 10

HOECHST Human

nuclei Hnuc/SOX2/SOX9

SOX2 SOX9 merge

WT OSCs on mEOC – Day 10

FM1-43 LIVE

Live imaging Surrounding cells Functional HC

SEM). Plutipotency genes (such as Nanog, Oct4 and Sox 2) which were highly expressed in WT hiPSCs were down-regulated in OSCs. In parallel the mRNA levels of otic markers such as PAX2, PAX8 and SIX1 significantly increased. We also observed a decreased expression of neurectoderm PAX6 and SOX1 genes when OSCs were placed in proliferative condition.

2

AS hiPSCs differentiated into AS otic progenitors Higher magnification of AS hiPSCs differentiated into AS OSCs

Hoechst SIX1 PAX2 SIX1/PAX2 Merge

10x 60x SOX9/Ki67 SOX9 Ki67 Hoechst SOX1 40x P4 P2

AS hiPSCs differentiated into AS OSCs

HOECHST MYOSIN6

hNUCLEI ATOH1

MYOSIN6/ATOH1 MERGE

Terminal differentiation into AS HC

AS OSCs P6 on mEOC – Day 10

Differenciation of AS hiPSCs into HCs

AS hiPSCs are able to differentiate into pathological otic progenitors similarly to WT hiPSCs, as shown by IF (3-A, 3-B) and qRT-PCR (3-C). It seemed that terminal differentiation into HCs was not affected by the ALMS1

mutation (3-D). Functional analyses are still in progress.

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Disease modeling

4

Reprogramming Otic induction Terminal differentiation

Modeling the Alstrom syndrome

Results obtained from the differentiation of AS hiPSCs into HCs suggest that the differentiation potential of these cells into cochlear cells is unaffected in AS patients. However, we observed some proliferation defects and increase in apoptosis between healthy and AS OSCs (4-A, 4-B).

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Isogenic hiPSCs

To be sure that observed phenotypes are only due to the ALMS1 disease-causing mutation, we needed isogenic cells. The strategy used to obtain them is described in panel 5-A. We used the CRISPR/Cas9 genome editing to correct the AS hiPSCs STOP mutation. The Cas9

binding is guided by base-pair

complementarities between the

engineered single-guide RNA (gRNA) and a genomic DNA target sequence. We designed gRNAs complementary to the ALMS1 genomic locus (5-B). Using the engineered gRNA, the Cas9 will induce specific double-stranded break (DSB), which can be repaired through the Homology Directed Repair (HDR) pathway. The HDR mechanism is induced by providing a single stranded

oligo dinucleotide (ssODN) repair

template to the AS hiPSCs. The ssODN contains a FokI site we will use to check homologous recombining events.

4D nucleofection

feeder-free pathological hiPSCs

antibiotic selection & clonal picking isogenic hiPSCs CRISPR/Cas9 components + Cas9-gRNA ssODN repair template

genomic PCR & FokI digestion sequencing

hiPSCs validation & characterization

5

4 Conclusion

We developed a human model to study the impact of ALMS1 mutation. We also performed genome

editing to obtain isogenic hiPSCs used as a reliable control. We obtained preliminary results indicating that

ALMS1 mutation does not affect differentiation of hiPSCs into HCs but induces proliferative defects and an

increase in apoptosis. Identification of molecular mechanisms implicated in these phenotypes is crucial to

understand the hearing loss in Alström syndrome.

Thanks to the University Medical Center Göttingen

which has kindly funded my bursary and offers me the opportunity to attend the Molecular Biology of Hearing and Deafness congress.

hiPSCs AS G1608A

hiPSCs AS 707CVD hiPSCs AS F2 hiPSCs WT

Hoechst NANOG ALMS1 Hoechst/ALMS1

Higher magnification of hiPSCs

1-B

1-C

Teratoma formation after injection in nude mice

Ectoderm _Mesoderm _Endoderm

1-D

2-A

2-B

2-C

2-D 2-E

Pluripotency Otic progenitor Neurectoderm

2-F 2-G 2-H 3-A 3-C 3-B 3-D 5-A 5-B AS OSCs P7

HOECHST SIX1 Ki67 pHH3 Ki67/pHH3

WT OSCs P7

HOECHST SIX1 Ki67 pHH3 Ki67/pHH3

10x 10x

HOECHST Ncadherin _SIX1 Ncad/HOECHST

AS OSCs P7 40x

HOECHST Ncadherin SIX1 Ncad/HOECHST

WT OSCs P7 40x

ciliogenesis proliferation,

cell cycle &

apoptosis fibrosis

AS OSC 4-A _{Proliferation of WT and AS OSCs}

HOECHST Caspase3* MYOSIN6/

Caspase3* HOECHST/ Caspase3*

AS HC 40x

MYOSIN6

HOECHST MYOSIN6 AcTUBULIN hNUCLEI Merge

WT HC 40x

Pathological HCs

ciliogenesis proliferation,

cell cycle & apoptosis

fibrosis

4-B

References

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• Jagger D., G. Collin, J. Kelly, E. Towers, G. Nevill, C. Longo-Guess, J. Benson, K. Halsey, D. Dolan, J. Marshall, J. Naggert, A. Forge. 2011. Alstrom syndrome protein ALMS1 localizes to basal bodies of cochlear hair cells and regulates cilium-dependent planar cell polarity. Human molecular genetics 20:466-481

• Oshima K., K. Shin, M. Diensthuber, A.W. Peng, A.J. Riccu, S. Heller. 2010. Mechanosensitive hair cell-like cells from embryonic and induced pluripotent stem cells. Cell. 141:704-716 • Zaluto E., F. Favaretto, C. Veronese, S. Camparano, J.D. Marshall, S. Romano, A. Cabrelle, G.B. Collin, B. Zavan, A.S. Belloni, E. Rampazzo, J.K. Naggert, G. Abatangelo, N. Sicolo, P.Maffei,