Background & Aims

Fibrodysplasia Ossificans Progressiva (FOP) is a rare and devastating genetic disorder characterized by progressive heterotopic ossification (HO) caused by gain of function mutation to the ACVR1 gene. Alongside its well-documented skeletal anomalies, individuals affected by FOP often endure another silent and persistent adversary: excruciating pain. This pain, which has recently been identified as largely neuropathic1, underscores the need to dissect the intricate interplay between the molecular dysregulations driving FOP and the complex nociceptive mechanisms orchestrating pain in this population. Induced pluripotent stem cell (iPSC) technology offers an innovative tool to studying pain in this population by enabling the generation of patient-derived sensory neurons in a controlled laboratory environment, bypassing the need for invasive procedures and minimizing the risk of exacerbating FOP symptoms.

Methods

In this ongoing study, peripheral blood mononuclear cells were collected from four patients with FOP. hiPSC lines will be generated using episomal vectors, sub-banked as a resource for the FOP research community and then differentiated using a novel 7 day directed differentiation protocol composed of daily combinations of small molecules and growth factors to recapitulate sensory neuron developmental stages2. Initially, single cell seeding density titrations will be carried out in 6-well plates to establish the best density for optimal differentiation before sensory neuron production will be scaled up in flask format and banked. Sensory neurons will then be thawed and matured before being molecularly and functionally characterized.

Results

PBMCs from one donor have been successfully reprogrammed into hiPSCs and sub-banked. Sensory neurons have been generated from this line and express ISLET1, BRN3A, PRPH, and TUJ1 at a purity of greater than 95% via immunocytochemistry. The three other donor lines have been transfected and colonies are expected to emerge in the coming weeks. The sensory neurons from the four different donor lines will be tested for similarities in gene expression profile via qPCR for the pan-sensory neuron ion channels: NaV1.7, NaV1.8, NaV1.9, TRPV1, CAV3.2, P2RX3, and TRKA. Lastly, sensory neurons derived from each line will be matured on multi-electrode array plates to look at functional differences in baseline activity through 4 weeks.

Conclusions

Results from this study will demonstrate that a rapid seven day directed differentiation protocol can be applied to multiple FOP patient hiPSC lines to successfully produce functional, sensory neurons for downstream studies to investigate pain in FOP and the role of ACVR1 in this pathology.

References

Yu X, Ton AN, Niu Z, Morales BM, Chen J, Braz J, Lai MH, Barruet E, Liu H, Cheung K, Ali S, Chan T, Bigay K, Ho J, Nikolli I, Hansberry S, Wentworth K, Kriegstein A, Basbaum A, Hsiao EC (2023) ACVR1-activating mutation causes neuropathic pain and sensory neuron hyperexcitability in humans. Pain 164:43-58.

Kalia AK, Rosseler C, Granja-Vazquez R, Ahmad A, Pancrazio JJ, Neureiter A, Zhang M, Sauter D, Vetter I, Andersson A, Dussor G, Price TJ, Kolber BJ, Truong V, Walsh P, Lampert A (2023) How to differentiate induced pluripotent stem cells into sensory neurons for disease modelling: a comparison of two protocols. Res Sq.

Presenting Author

Vincent Truong

Poster Authors

Vincent Truong

Anatomic Incorporated

Lead Author

E-mail

Patrick Walsh

MS

Anatomic Incorporated

Lead Author

E-mail

Hajira Elahi

PhD

University of Texas at Dallas

Lead Author

E-mail

Daniel Perrien

PhD

Emory University

Lead Author

E-mail

Theodore Price

PhD

University of Texas at Dallas

Lead Author

E-mail

Topics

  • Models: Chronic Pain - Neuropathic