Background & Aims
Pain following spinal cord injury (SCI) is clinically challenging. Transplantation of chromaffin cells in the spinal subarachnoid space has shown chronic pain reduction in preclinical models and pilot clinical work [1]. However, sufficient donor cell sources have been limited. The use of human iPSCs to generate human chromaffin cells (hCCs) from skin fibroblasts can overcome this limitation. To further boost antinociceptive potency, grafted chromaffin cells could be engineered to produce supplementary pain-reducing neuroactive substances. In particular, the N-type Ca2+ channel blocker, omega conopeptide MVIIA has been effective in several pain models [2]. NMDA antagonist, serine-histogranin (SHG) shows inhibitory properties at NMDA receptors in both in vitro and in vivo models [3]. The goal of the current study is to optimize parameters for skin-derived hCC transplants in the spinal subarachnoid space and to generate recombinant hCCs to further boost reduction of SCI chronic pain.
Methods
Skin fibroblast-derived hiPSCs from male and female donors were reprogrammed using integration-free Sendai and confirmed for pluripotency. Cells were differentiated to hCCs following the protocol of Abu-Bonsrah et al 2018 [3]. Recombinant cells were engineered using AAV2/8 vector encoding MVIIA and multimers of 6SHG previously generated by our lab. Cell cultures were immunostained to confirm the presence of differentiated chromaffin cell phenotypes and expression of MVIIA and SHG. Male and female Sprague Dawley rats underwent spinal cord injury (SCI) by 60 second clip compression at T7 [4]. Sex-matched hCC lines (50-80K) were intrathecally transplanted in Vitrogel 4 weeks post-SCI to lumbar spinal levels. Tactile, cold and heat hypersensitivity were assessed by standard behavioral tests at baseline and weekly thereafter post-injury. Transplants were evaluated for survival and expression of chromaffin cell and recombinant peptide markers by immunocytochemistry and FLISA/ELISA.
Results
All skin cell lines were successfully differentiated into the chromaffin phenotype. The presence of chromaffin cell markers and the release of catecholamines were compared between different cell lines, with comparable outcomes between lines of age-matched male and female donors. Intrathecal injection of hCCs in Vitrogel led to reduced pain-related hypersensitivity in SCI animals on all 3 outcome measures compared with controls. Recombinant MVIIA and MVIIA/6SHG hCCs were generated and the presence and secretion of MVIIA and SHG peptides confirmed in cells and in culture media. Intrathecal delivery of the recombinant cells further potentiated the antinociceptive effects of the hCCc, with particularly strong outcomes in the MVIIA/6SHG group. Intrathecal Injection of MVIIA and SHG antibodies transiently attenuated the observed analgesic effect of the recombinant grafts. The presence of recombinant peptides in the spinal tissue and grafted hCCs was confirmed by FLISA and immunostaining.
Conclusions
Skin fibroblast derived human iPSCs from various donors can be induced to a chromaffin cell phenotype expressing chromaffin cell markers and catecholamine secretion. Intrathecal transplantation of hiPSC-derived chromaffin cells (hCCs) reduces SCI neuropathic pain symptoms for at least 8 weeks following transplantation. Transduction of hCCs with additional complementary analgesic peptides such as MVIIA and SHG may further boost SCI pain reduction. Together, these studies support the development of skin fibroblast hiPSC derived chromaffin cell intrathecal transplantation for the management of SCI pain.
References
1.Sagen J, Hernandez M, Eeswara A, Gross S, Jergova S. Chapter 19 – Cell transplantation for reducing neuropathic pain after SCI. In: Sang CN, Hulsebosch CE, editors. Spinal Cord Injury Pain: Academic Press; 2022. p. 389-421.
2.Hama A, Sagen J. Antinociceptive effects of the marine snail peptides conantokin-G and conotoxin MVIIA alone and in combination in rat models of pain. Neuropharmacology. 2009;56(2):556-63. doi: 10.1016/j.neuropharm.2008.10.008. PubMed PMID: 19010337; PMCID: 2735251. https://www.ncbi.nlm.nih.gov/pubmed/19010337
3.Hentall ID, Hargraves WA, Sagen J. Inhibition by the chromaffin cell-derived peptide serine-histogranin in the rat’s dorsal horn. Neurosci Lett. 2007;419(1):88-92. Epub 2007/04/20. doi: S0304-3940(07)00380-1 [pii]
10.1016/j.neulet.2007.03.056. PubMed PMID: 17442490; PMCID: 1945824. https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17442490
4.Abu-Bonsrah KD, Zhang D, Bjorksten AR, Dottori M, Newgreen DF. Generation of adrenal chromaffin-like cells from human pluripotent stem cells. Stem cell reports. 2018;10(1):134-50. Epub 2017/12/14. doi: 10.1016/j.stemcr.2017.11.003. PubMed PMID: 29233551; PMCID: PMC5768882. https://www.ncbi.nlm.nih.gov/pubmed/29233551
5.Bruce JC, Oatway MA, Weaver LC. Chronic pain after clip-compression injury of the rat spinal cord. Exp Neurol. 2002;178(1):33-48. Epub 2002/12/04. doi: 10.1006/exnr.2002.8026. PubMed PMID: 12460606.
The University of Miami and J.S. and S.J. hold rights to intellectual property used in the study and may financially benefit from the commercialization of the intellectual property.
Supported by the Craig H. Nielson Foundation SCIRTS 890307.
Presenting Author
Jacqueline Sagen
Poster Authors
Jacqueline Sagen
PhD
University of Miami School of Medicine
Lead Author
Lauren Kelly Tierney
University of Miami, Miller School of Medicine, Miami, Florida, USA
Lead Author
Vama Shah
University of Miami, Miller School of Medicine, Miami, Florida, USA
Lead Author
Anjalika Eeswara
University of Miami, Miller School of Medicine, Miami, Florida, USA
Lead Author
Kristin Perrucci
University of Miami, Miller School of Medicine, Miami, Florida, USA
Lead Author
Stanislava Jergova
Univ of Miami
Lead Author
Topics
- Specific Pain Conditions/Pain in Specific Populations: Neuropathic Pain - Central