Successful Transplantation of Friedreich Ataxia Induced Pluripotent Stem Cell (iPSC)-Derived Sensory Neurons in Dorsal Root Ganglia of Adult Rodents

Jamshed Arslan, Pharm D, PhD

The dorsal root ganglia (DRG) are a collection of cell bodies of sensory nerves carrying sensory information – including nociception, mechanoreception and proprioception – from peripheral nervous system (PNS) to the central nervous system (CNS). Degeneration of DRG, as observed in the autosomal recessive disease Friedreich ataxia (FRDA), manifests physiologically as impaired speech, difficulty walking, and loss of sensation in arms and legs. To mitigate FRDA, cell replacement therapy (CRT), such as human pluripotent stem cells (hPSCs) or induced pluripotent stem cells (iPSCs) transplantation, and gene therapy, are emerging as viable therapeutic approaches. Previously, a team of researchers in Australia led by Drs. Mirella Dottori and Lachlan Thompson, provided the first evidence of long-term survival and integration of FRDA iPSC-derived neural progenitors into the cerebellum of adult rats. Now, Drs. Dottori and Thompson took their studies to the next level and grafted cells in the technically challenging DRG area. The transplanted hPSCs and FDRA iPSCs successfully differentiated into neuronal and glial cells in adult rodents, and satisfactorily expressed the markers for sensory neuronal subtypes of DRG.

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Characterizing Cells Before the Transplant

The research team used two cell lines for transplantation: hPSC and FRDA iPSC; the latter of which was generated by reprogramming FRDA patient-derived fibroblasts. The stem cells were then analyzed for the presence of markers of sensory neurons of DRG through RT-qPCR and immunostaining. Table 1 describes key features of the donor cells for transplantation.

Table 1: Characteristics of the donor cells.

A higher RNA expression of genes associated with nociceptors and mechanoreceptors was reported at week-3 of differentiation of FRDA cells: PLXNC1, TAC1 and VGLUT3 (nociceptive neurons), CALB1 (mechanoreceptive neurons), and RET (both nociceptors and mechanoreceptors). Concomitantly, a non-significant decrease in the expression of proprioceptive markers (PV and SPP1), was observed, which, according to the authors, alluded to the neurodegeneration associated with FRDA.

In other words, the cells selected for transplantation showed a profile consistent with DRG neurons. The next step was to transplant these human-derived cells into the rodent models of FRDA.

Immunofluorescence image showing Tyrosine Hydroxylase (TH) expression using Mouse Anti-Tyrosine Hydroxylase Monoclonal Antibody (779427) (MAB7566)in mouse ESCs differentiated into dopaminergic neurons. Following the TH primary antibody, cells were stained with Northern­Lights™ 557-conjugated Anti-IgG Secondary Antibody (NL007) (red). Cells were additionally stained with Mouse Anti-Neuron-specific beta-III Tubulin Monoclonal Antibody (TuJ-1) (NL1195V) conjugated to Northern­Lights™ 637 (white). DAPI was used for counterstaining cell nuclei (blue). Staining results reveal that TH expression is specifically localized to the cytoplasm of dopaminergic neurons.

Characterizing Cells After the Transplant

To explore how the donor cells survive and differentiate inside the rodent DRG, sensory progenitors derived from reporter mCherry- or GFP-hESCs were transplanted into the DRG region of young rats. Two weeks after transplantation, animals were sacrificed to perform immunostaining and histological analyses of DRG. As expected, positive staining for human mitochondria, human nuclei, glial marker S100B, neuronal marker β3-tubulin (TUBB3), PRPH, and DRG neuronal subtype markers for nociception (TrkA), mechanoreception (TrkB), and proprioception (TrkC) was observed.

Successful transplantation of reporter hPSC lines, notwithstanding a few negligible instances of ectopic injection of donor cells, encouraged the team to study the long-term outcome of transplanting FRDA iPSC-derived cells. To do so, the researchers generated sensory neurospheres from GFP reporter FRDA iPSCs and transplanted them into the DRG of nude rats. This time, the analysis was performed at eight weeks following transplantation. As predicted, phenotypic characterization of grafted cells indicated all the necessary neuronal and glial markers, as well as specific markers of DRG cells. Positive markers included TUBB3, PRPH, TRPV1, TrkA, TrkB, TrkC, S100B, and GFAP. The team also found neuronal projections from the transplanted cells extending into the spinal cord and along the neuronal tracts.

In sum, the researchers demonstrated the feasibility of CRT in FRDA by experimentally transplanting neuronal progenitors into DRG, an area characteristically damaged in FRDA.

(Left) Fluorescent IHC staining showing TrkB expression in frozen sections of mouse spinal cord using Goat Anti-TrkB Polyclonal Antibody (AF1494) overnight and tissue was stained with the NorthernLights™ 557-conjugated IgG Secondary Antibody (NL001) (red) and the nuclei were counterstained with DAPI (blue). TrkB staining was specific to both neuronal processes and cell bodies. (Right) Immunohistochemical analysis of Calbindin D detection in immersion fixed paraffin-embedded sections of human hippocampus tissue using Goat Anti- Calbindin D Polyclonal Antibody (AF3320) following by IgG Secondary Antibody [HRP Polymer] (VC004). The brain tissue sections were stained with DAB (brown) and counterstained with hematoxylin (blue). Specific staining for Calbindin D was localized to the synaptic vesicles and neuronal cell bodies of the hippocampus.

Significance

The successful transplant of human sensory progenitor cells into the DRG of adult mammals is a phenomenal step forward for the field of field of CRT. Future studies can build on this research by contrasting the in vitro and in vivo expression of neuronal and glial markers in the transplanted cells after prolonged timepoints. The response to sensory stimuli in a greater number of transplanted animals and cell lines also needs to be established. This research can pave the way for CRT in human PNS degeneration, especially the DRG neuropathies like FRDA.

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Note:
Viventi el al. used Goat Anti-Calbindin D/S100G Polyclonal Antibody (AF3320), Mouse Anti-Plexin C1 Monoclonal Antibody (MAB3887), Sheep Anti-Parvalbumin alpha Polyclonal Antibody (AF5058), Goat Anti-Osteopontin/OPN/SSP1 Polyclonal Antibody (AF1433), Rabbit Anti-TRPV1 Polyclonal Antibody (NB120-3487), Goat Anti-TrkA Polyclonal Antibody (AF175), Mouse Anti-TrkB Monoclonal Antibody (NBP1-47898), Goat Anti-TrkB Polyclonal Antibody (AF1494), Rabbit Anti-Tyrosine Hydroxylase (NB300-109), and BMP2 (R&D Systems, 10 ng/mL).

Jamshed Arslan, Pharm D, PhD   
Dr Arslan is an Assistant Professor at Salim Habib University (formerly, Barrett Hodgson University), Pakistan. His interest lies in neuropharmacology and preparing future pharmacists.

Research in focus

Viventi, S., Frausin, S., Howden, S. E., Lim, S. Y., Finol-Urdaneta, R. K., McArthur, J. R., Abu-Bonsrah, K. D., Ng, W., Ivanusic, J., Thompson, L., & Dottori, M. (2021). In vivo survival and differentiation of Friedreich ataxia iPSC-derived sensory neurons transplanted in the adult dorsal root ganglia. Stem cells translational medicine, 10(8), 1157–1169. https://doi.org/10.1002/sctm.20-0334