Background & Aims

Chronic neuropathic pain following spinal cord injury (SCI) significantly reduces quality of life and impedes rehabilitation. Prolonged delivery of analgesic substance release by highly secretory chromaffin cells showed promising outcomes for chronic pain in preclinical models [1]. Advances in stem cell technologies allow for generation of autologous sources of chromaffin cells using iPSCs. Effects of grafted cells may be potentiated by genetically engineering additional cDNAs of potent analgesic peptides. Previous studies showed beneficial effects of recombinant NMDA antagonist serine-histogranin (SHG) or combination of SHG with opioid peptide endomorphin 1 (EM1) on SCI pain [2]. The aims of the present study were i) to evaluate capacity of blood-derived hiPSCs from different donors to differentiate into chromaffin cells, ii) to engineer hiPSC-derived chromaffin cells (hCCs) producing SHG and SHG-EM1 and iii) to evaluate their analgesic effect in an SCI model of chronic pain in rats.

Methods

hiPSCs derived from human peripheral blood were differentiated to hCCs using a series of factors according to Abu-Bonsrah et al. (2018) protocols [3]. Cultures were confirmed for production of chromaffin cell markers and catecholamines. To generate recombinant cells, AAV8/2 viral particles encoding 6SHG and 6SHGEM1 were added to differentiated cells. Cells were fixed with 4% paraformaldehyde and processed for standard ICC staining. Media was collected and processed for ELISA evaluation of catecholamines. Male rats (Sprague Dawley, 120g-140g) underwent SCI by 60 sec clip compression at T8 [4]. Cells were mixed with Matrigel (80-150k in 5 µl-10µl) and transplanted intrathecally; rats were immunosuppressed. Standard tests for tactile, cold and heat hypersensitivity were used. CSF and fresh lumbar spinal tissue were used for ELISA/FLISA analysis for the presence of catecholamines and recombinant peptides.

Results

Each tested cell line was successfully differentiated into chromaffin phenotypes. Cell lines from older donors had a lower yield of chromaffin cells and markers. Cell densities of 80-150K in 3-5mg/ml of Matrigel showed the most optimal results for chromaffin cell survival. Intrathecal injection of each cell line (80-100K) provided reduction in chronic pain behavior in SCI animals. The cells from older donors had lower survival and longevity after grafting. All tested cell lines were suitable for engineering of recombinant cells. 6SHG and 6SHGEM1rRecombinant cells showed higher analgesic potency compared to the nonrecombinant cohorts, including cells lines from older donors. Intrathecal injection of an SHG antibody reduced the effects of recombinant cells. The presence of recombinant peptides SHG and EM1 in transplanted spinal cords were confirmed by FLISA.

Conclusions

Blood-derived hiPSCs of various backgrounds can be differentiated to >70% chromaffin-like cells producing catecholamines indicating good consistency and potential generalizability of the differentiation protocol for future autologously derived cells for transplantation. A possible exception may be blood-derived hiPSCs from elderly donors. hCCs transplanted in the lumbar spinal subarachnoid space attenuated pain-related symptoms following spinal cord injury. Successful transplants resulted in increased spinal catecholamine levels. hCCs can be genetically enhanced to produce supplementary analgesic peptides serine-histogranin and endomorphin-1 Recombinant hCCs appeared to boost the analgesic effects of the intrathecal grafts on SCI pain. These preliminary findings suggest that intrathecally transplanted hiPSC-derived chromaffin cells may provide a relatively non-invasive approach for the management of chronic 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.Jergova S, Gordon CE, Gajavelli S, Sagen J. Experimental gene therapy with serine-histogranin and endomorphin 1 for the treatment of chronic neuropathic pain. Frontiers in molecular neuroscience. 2017;10:406. Epub 2017/12/26. doi: 10.3389/fnmol.2017.00406. PubMed PMID: 29276474; PMCID: PMC5727090. https://www.ncbi.nlm.nih.gov/pubmed/29276474
3.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
4.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 DOD SC210210

Presenting Author

Stanislava Jergova

Poster Authors

Stanislava Jergova

PhD

Univ of Miami

Lead Author

E-mail

Anjalika Eeswara

University of Miami, Miller School of Medicine, Miami, Florida, USA

Lead Author

E-mail

Kristin Perrucci

University of Miami, Miller School of Medicine, Miami, Florida, USA

Lead Author

E-mail

Greta Servadei

University of Miami, Miller School of Medicine, Miami, Florida, USA

Lead Author

E-mail

Holly Cukier

University of Miami, Miller School of Medicine, Miami, Florida, USA

Lead Author

E-mail

Jacqueline Sagen

University of Miami School of Medicine

Lead Author

E-mail

Topics

  • Specific Pain Conditions/Pain in Specific Populations: Neuropathic Pain - Central