In vivo survival and differentiation of Friedreich ataxia iPSC‐derived sensory neurons transplanted in the adult dorsal root ganglia

Abstract Friedreich ataxia (FRDA) is an autosomal recessive disease characterized by degeneration of dorsal root ganglia (DRG) sensory neurons, which is due to low levels of the mitochondrial protein Frataxin. To explore cell replacement therapies as a possible approach to treat FRDA, we examined transplantation of sensory neural progenitors derived from human embryonic stem cells (hESC) and FRDA induced pluripotent stem cells (iPSC) into adult rodent DRG regions. Our data showed survival and differentiation of hESC and FRDA iPSC‐derived progenitors in the DRG 2 and 8 weeks post‐transplantation, respectively. Donor cells expressed neuronal markers, including sensory and glial markers, demonstrating differentiation to these lineages. These results are novel and a highly significant first step in showing the possibility of using stem cells as a cell replacement therapy to treat DRG neurodegeneration in FRDA as well as other peripheral neuropathies.


| INTRODUCTION
The peripheral nervous system (PNS) is one of the primary and most significant sites of degeneration occurring in Friedreich ataxia (FRDA).
FRDA is an autosomal recessive disease and it is considered the most common form of all inherited ataxias known to date. 1  expressed and, within the mitochondria, helps the formation of ironsulfur cluster functioning as an iron-chaperone. 4 Low levels of FXN lead to a reduction in iron-sulfur cluster synthesis with concomitant mitochondrial iron accumulation, mitochondria dysfunction, as well as an increased cellular oxidative stress. 5,6 All these events cause cell toxicity and cell death, particularly within the nervous system and cardiac tissue. Large sensory DRG neurons and cerebellar neurons are mainly affected within the nervous system. However, as clinical assessments are becoming more sensitive, several other neurological pathways appear to be affected, including the auditory and visual systems as well as speech. [7][8][9] These findings suggest that neurodegeneration occurs within multiple neuronal cell types.
Although many breakthroughs have been made in unveiling some pathological mechanisms of the disease, others are still unclear. To date, most therapies are only aimed at slowing down the degenerative process and/or managing the symptoms. Several clinical trials are ongoing together with new studies underway to evaluate alternative future interventions. 10,11 In particular, gene therapy has attracted much interest thanks to its successful application in treating other disorders. [12][13][14][15] While drug and gene therapies are progressing to treat neurodegeneration and cardiomyopathy occurring in FRDA, cell replacement therapy remains an attractive treatment strategy to replenish some of the most severely affected mutant FXN cells.
Many transplantation studies have successfully demonstrated the ability of human donor cells to survive and functionally integrate in the central nervous system (CNS) leading to damaged neural tissue repair. 16 Many of these studies utilized human fetal tissue, however, ethical concerns prompted exploration of alternative sources, particularly human pluripotent stem cells (hPSCs). Indeed, both mouse and human PSCs differentiated to neurons and transplanted in Parkinsonian rats showed comparable therapeutic potential. [17][18][19] Transplantation of hPSC-cell derivatives has also been examined for other neurodegenerative conditions and neurological disorders, such as animal models of epilepsy, [20][21][22][23] and spinal cord injury, 24 showed expression for β3-tubulin at 1 month post-injection; however, no neuronal cells were observed in the long-term study. Similar results were observed after transplantation of dissociated neural stem cells in the intact DRG. 29 Other studies showed transplantation of mesenchymal stem cells into the DRG for investigating their therapeutic potential to treat pain through release of cytokines and chemokines. 30,31 In 2015, Hoeber et al analyzed the recovery of sensorimotor functions after dorsal root avulsion in mice. 32 They placed hESC-derived neural progenitor spheres along the spinal cord with access to the avulsed areas of L3-L5 spinal cord. Immunostaining revealed that transplanted human neural progenitors differentiated to neurons and glia. However, donor-derived neurons did not show connections with the intrinsic spinal cord circuitries because they were unable to reach that region. Nevertheless, improvement in the animal's sensorimotor functionality was observed in behavioral functional tests, suggesting partial regeneration of sensory innervation into the spinal cord. Similar studies have been performed using rat boundary cap neural crest stem cells (bNCSCs) whereby they were placed in the dorsal root-spinal cord junction following dorsal root avulsion injury. 33 The presence of transplanted cells in the proximal part of the dorsal root was observed along the dorsal regions of the spinal cord. In contrast, no donor cells were found in the spinal cord of transplanted animals with intact dorsal roots, suggesting that the migration of bNCSCs occurs in response to injured tissue.
Earlier work from our laboratory has provided the only evidence of cell replacement therapy to treat FRDA available to date. 34  showed that FRDA iPSC-derived neural progenitors transplanted into the cerebellum of adult rats survived, integrated, and differentiated within the host tissue. Grafted cells expressed neuronal markers NeuN and Tbr1, the glial marker GFAP, as well as markers of immature and mature oligodendrocytes, Olig2 and APC, respectively. These results are consistent with long-term survival and integration of FRDA iPSC-neuronal derivatives in the adult nervous system.   Figure S3D).

| Culture and differentiation of hESCs and FRDA iPSCs
Human ES cell line, H9, and human FRDA iPSC cell line, FA10, were maintained as bulk culture in feeder-free conditions on vitronectin (StemCell Technologies) coated flasks (Corning) using TeSR-E8 basal medium (StemCell Technologies). Both cell lines were differentiated following the protocol for sensory differentiation as previously described. [36][37][38] For induction, both H9 hESC and FA10 iPSC cell lines were plated onto freshly laminin (Invitrogen)-coated organ culture plates and cell culture media changed every 2 days. On the fifth day, cells were enzymatically detached using 0.5 mM EDTA (Life Technologies) and cultured in suspension. The generated NSPs were cultured for further 5 days, supplemented with basic fibroblast growth factor 2 (FGF2) (20 ng/mL, Peprotech) and BMP2 (10 ng/mL, R&D system) to form NSPs consisting of neural crest progenitors using the published protocol. 36

| Statistical analysis
Statistical analysis was performed using GraphPad Prism 8 software and data were presented as mean with error bars representing SE of mean (SEM). Statistical significance was evaluated using independent groups (unpaired) two-tailed t-tests for expression of each marker at 1 and 3 weeks of differentiation in both hESC and FRDAderived sensory neurons.

| Animals
The Animals were housed in individually ventilated cages on a 12 hours light/dark cycle with ad libitum access to food and water.
Rats aged ≥10 weeks, both Sprague-Dawley and athymic strains, were included in the study. Females were used over males for housing purposes, given there is no evidence to suggest any sex-effect on the outcome of the experiment.

| Surgical procedure
The surgical procedures were done using a stereotaxic frame for mCherry, n = 6 for FA10-GFP cell line, respectively) 6 weeks, and 8 weeks (n = 3 for FA10-GFP cell line).

| Pharmacological immunosuppression
Injectable immunosuppressant preparations were made fresh and kept at 4 C for 1 week. Cyclosporin A (Labseeker) was first dissolved in absolute ethanol and then emulsified in olive oil. Immunosuppressant was administered daily (10 mg/kg) starting from 5 days prior to surgery. Immunosuppressant was administered to animals by subcutaneous injection. Treatment with the compound continued for the duration of the study.

| Immunostaining and imaging
For immunostaining, cells cultured on coverslips were fixed with 4% PFA on ice for 10 minutes. Cells and DRG tissues were permeabilized for 15 minutes at room temperature using 0.2% triton-X100 solution.
Incubation with primary and secondary antibodies was performed in  Immunostaining analyses were conducted to determine the cellular identities of the hESC-derived donor cells. HESC-derived cells showed positive staining for the neuronal marker β3-tubulin (TUBB3) ( Figure 3B) as well as the glial marker S100β ( Figure 4B). Transplanted  Figure 5). Note that some donor cells may co-express TRK receptors during early stages of differentiation, as described in DRG development. 40 The donor neurons showed bipolar elongated processes, as expected for embryonic DRG sensory neurons ( Figure 3C). 41 One technical challenge of transplanting cells into the DRG is that the DRG is surrounded by connective tissue, which makes the penetration of the DRG using a glass capillary difficult. Instances of ectopically injected cells outside the DRG are documented in Figure 4. In  Figure 7C). In addition to neuronal markers, donor FA10-GFP + cells expressing glial markers, S100β and GFAP, were also detected ( Figure 6E,F). Interestingly, human cells positive for the S100β marker surrounded the host sensory neurons ( Figure 6E) similarly to that which occurs with endogenous S100β + satellite glial cells. 42   One of the primary and most severe sites of neurodegeneration that is consistently observed in FRDA is the DRG. [45][46][47] However, neurodegeneration also occurs in other neuronal populations within the CNS and PNS and for some patients, degeneration is also observed in non-neuronal tissues such as cardiac tissue. 48 For this reason, it is considered that a combination of multiple therapeutic interventions may be needed to ameliorate

CONFLICT OF INTEREST
The authors declared no potential conflicts of interest.

AUTHOR CONTRIBUTIONS
S.V.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; S.F.: conception and design, collection and/or assembly of data, data anal- conception and design, financial support, administrative support, provision of study material, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.