By Sashini Ranawana
It is often easy to take for granted the ability to detect sensory stimuli and bring about voluntary movement. This, however, is not an option for individuals suffering from any one of the numerous forms of paralysis, where the disruption of neuronal signalling between the central nervous system (CNS) and peripheral nervous system (PNS) is accompanied by impaired sensation and motor control.1 Such a loss of function can be brought about by a multitude of causes: spinal cord injuries that affect all motor and sensory neurons at spinal levels below the site of damage; medical conditions such as amyotrophic lateral sclerosis and stroke; and hereditary disorders such as hereditary spastic paraplegia.1,2 The manifestations of paralysis are as varied as its causes, but always involve to some extent, reduced muscle strength, which is sometimes associated with a decreased ability to sense signals from the external environment. Ultimately, this reduces the quality of a patient’s life.
Regaining full or even partial nervous function after paralysis is rare, but experiments of new potential regenerative therapies hold promise. These approaches aim to produce healthy neurons to replace damaged ones, through the controlled differentiation of human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells.4,5,6 Theoretically, these neurons could allow signals to pass from the CNS to the PNS, when transferred back to the affected region of the nervous system. Until recently the focus has been on generating motor neurons,4,5 but new protocols are now being developed for dorsal sensory neurons as well.6 When transplanted together, they could potentially re-enable both movement and feeling.
The isolation of ES cells from human pre-implantation blastocysts is an ethical implication that has limited their clinical applications. iPS cells are one way to circumvent this problem. They can be derived through the undifferentiation of somatic cells and can be similarly directed to differentiate, due to their pluripotent nature, into any cell type. Using these patient-specific cells in an autologous transplant provides a clear benefit by eliminating the need for immunosuppressive drugs.
In a study conducted by Li et al. in 2005, mature spinal motor neurons were produced from ES cell-derived neuroepithelial stem cells.4 This involved treating cells with retinoic acid at an early stage of development, before they expressed the Sox1 transcription factor. By culturing these stem cells on laminin in the presence of the sonic hedgehog (SHH) glycoprotein, cells expressing the motor neuron marker HB9 were eventually detected. A similar study was performed by Karumbayaram et al. in 2010, with human iPS cells.5 These cells were grown into three dimensional embryoid bodies before treatment with retinoic acid and the SHH-mimicking molecule, purmorphamine. In both studies, functional motor neurons with the ability to transmit electrical signals, were produced. These in vitro-generated excitable cells have great therapeutic potential in vivo.
Neurogenesis requires specific combinations of extracellular signalling molecules, at particular developmental stages and concentrations. Therefore, developing differentiation protocols for various subsets of neurons is challenging. By 2018 however, researchers at the University of California, Los Angeles, had developed an efficient method to derive spinal sensory neurons from human pluripotent cells, expanding on what was already known about producing motor neurons.6 Dorsal sensory neurons arise at different times, in the presence of distinct biomolecules, compared to ventral motor neurons. By analysing the expression of particular transcription factors at various stages, researchers were able to confirm the movement of ES cells and iPS cells towards the sensory lineage. Transcription levels of Pax3, a dorsal spinal progenitor marker, increased after exposure of neuroepithelial cells to retinoic acid. This was followed by treatment with Bone Morphogenic Protein 4, a molecule secreted by dorsal roof plate cells of the neural tube during neurogenesis in vivo. It was found that sensory dorsal interneurons (dI)1 and dI3 were formed with a differentiation efficiency of 35%. The DCC and ROBO3 receptors expressed on these maturing neurons indicated that they were functional and were capable of elongating their axons to form synapses. Sensory systems are crucial for perceiving stimuli such as pain and temperature, for reflex actions in response to dangerous environmental triggers and for controlling appropriate movements. Moreover, both afferent and efferent pathways of the nervous system are affected in many cases of paralysis. It is clear that re-establishing feeling is as important as restoring muscle function in patients with this degenerative condition.
The possibilies of pluripotent stem cells are endless, and with the development of iPS cell technology, it is now possible to generate cells that won’t be rejected by a patient. Nevertheless, major hurdles still have to be overcome before transplants of neurons can become routine procedures. With ES cells and iPS cells, the efficiency of differentiation is one of the most important considerations. Since these cells have the potential to develop into all cell types upon exposure to different molecules present in the extracellular environment, they can easily form tumours when transplanted into the tissues of organism.7 Even a single cell amongst terminally differentiated neurons has this tumorigenic potential. Thus, only pure populations of mature neuronal cells can be selected for therapeutic use in vivo. In addition, regenerative cell transplants are most effective when performed shortly after tissue damage occurs.8 Patient-specific cells often take long to produce, by which time scar formation might have limited their efficacy for tissue repair. The question of how a sufficient number of cells can be generated efficiently and rapidly needs to be prioritized when it comes to stem cell therapy.
For the moment, these dorsal sensory and ventral motor neurons can be used for disease modelling in vitro. This might facilitate drug screening before pre-clinical trials and can help scientists obtain a better understanding of how specific neurological diseases develop in patients. However, it is likely that these cells will soon be used for treatment purposes as well. These neuron differentiation protocols will be further developed and refined, and it is possible that paralysis, an untreatable condition now, might one day be cured.
(1) NHS. Paralysis. Available from: https://www.nhs.uk/conditions/paralysis/ [Accessed 24 January 2021].
(2) National Institute of Neurological Disorders and Stroke. Spinal Cord Injury: Hope Through Research. Available from: https://www.ninds.nih.gov/disorders/patient-caregiver-education/hope-through-research/spinal-cord-injury-hope-through-research [Accessed 25 January 2021].
(3) National Center for Advancing Translational Sciences. Hereditary spastic paraplegia. Available from: https://rarediseases.info.nih.gov/diseases/6637/hereditary-spastic-paraplegia [Accessed 27 January 2021].
(4) Li X, Du Z, Zarnowska E W, Pankratz M, Hansen L O, Pearce R A, et al. Specification of motoneurons from human embryonic stem cells. Nature Biotechnology. 2005;23(2): 215-221. Available from: doi: 10.1038/nbt1063.
(5) Karumbayaram S, Novitch B G, Patterson M, Umbach J A, Richter L, Lindgren A, et al. Directed differentiation of human induced pluripotent stem cells generates active motor neurons. Stem Cells. 2009;27(4): 806-811. Available from: doi: 10.1002/stem.31.
(6) Gupta S, Sivalingam D, Hain S, Makkar C, Sosa E, Clark A, et al. Deriving Dorsal Spinal Sensory Interneurons from Human Pluripotent Stem Cells. Stem Cell Reports. 2018;10(2): 390-405. Available from: doi: 10.1016/j.stemcr.2017.12.012
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(8) EuroStemCell. Spinal cord injuries: how could stem cells help?. Available from: https://www.eurostemcell.org/spinal-cord-injuries-how-could-stem-cells-help [Accessed 28 January 2021].