Tissue-type plasminogen activator and human NPCs: two peas in one spinal cord

Written by RegMedNet

In this editorial, Pinar Mesci, Ph.D. (Assistant Project Scientist in Dr. Alysson Muotri’s laboratory, University of California San Diego) discusses her research on neural stem cell transplantations.

Read Pinar’s recent article, “Tissue-type plasminogen activator-primed human iPSC-derived neural progenitor cells promote motor recovery after severe spinal cord injury”, here>>

Stem cell therapy, initially limited to bone marrow transplants, has been a promising avenue in several fields of medicine, specifically in blood-related disorders. Since the first derivation of human embryonic stem cell (hESC) lines and the recent advances in induced pluripotent stem cell technology (or iPSC), we have now a unique opportunity to tackle and provide stem cell therapies for a wide range of diseases including patients with neurodegenerative disorders and brain or spinal cord injury.

Similar to other stem cell laboratories, the Muotri laboratory has been using hESCs and hiPSCs for the last decade to model neurodevelopmental disorders and to perform targeted drug screenings for disorders where animal models fell short in recapitulating the human disorder.

Recently, Professor Wendy Campana at University of California, San Diego (UCSD; CA, USA) approached us with a very interesting project regarding the pain management after spinal cord injury (SCI). Indeed, one of the most debilitating symptoms of SCI is pain caused by damaged nerve fibers in the spinal cord. Current treatment strategies involving stem cell transplantations are mostly limited to human umbilical cord tissue-derived mesenchymal stem cells (MSCs) which are under US Food and Drug Administration (FDA) regulation. Interestingly, another treatment approach has been tissue engineering and identifying or engineering new scaffolds that can not only help re-create and maintain tissue stability but could also be used to efficiently deliver potential treatments to the site of injury.

In this context, Dr Campana’s team tested tissue-type plasminogen activator (tPa), an activator of fibrinolysis and globally approved drug for treatment of certain types of strokes based on its protease function — to alleviate neuropathic pain following spinal cord injury. Under physiological conditions, pre-synaptic vesicles have been shown to contain tPa and tPa was shown to be secreted by neuronal growth cones and to promote synaptic plasticity. Interestingly, tPa expression was found to be increased in the cerebellum, specifically in Purkinje neurons during motor task training and motor learning. Non-proteolytic activities, such as NMDAR-dependent cell signaling, can be recapitulated by enzymatically-inactive (EI) tPa. But, the activity of tPa or of EI-tPa in hiPSCs or human iPS-derived neural precursor cells (hiNPCs) had not been studied.

Given the broad range of mechanisms of action of EI-tPa, Dr. Campana’s team hypothesized that EI-tPa-treated stem cell transplantation could improve not only the survival of the transplanted cells but could also alleviate the neuropathic pain caused by spinal cord injury. Thus, by combining a stem cell and scaffolding approach, they aimed to improve the stem cell transplantation conditions.

The Campana lab used hiNPCs obtained from a healthy donor, containing a genetically encoded GFP tag which we provided to them. In the Muotri lab, we have previously used these cells in many neurological disorders as they are capable of differentiating into cortical neurons or glial cells; however, in the absence of additional differentiation/patterning factors, their potential to giving rise to motor neurons was questionable.

The hiNPCs were first treated with EI-tPa prior to the transplantation to the site of injury. At first the results we obtained were disappointing, because we did not observe any improvement of the neuropathic pain. However, we did not observe a worsening of neuropathic pain either and the pain related genes were altered in the dorsal root ganglion (DRG) after 4 months, possibly indicating an improvement in sensory function in long term, which is important for safety and future translation to the clinic.

This is when things got interesting. The rats subjected to a thoracic spinal cord injury showed recovery of some motor functions, such as improved locomotor activity! When we examined closely the faith of the transplanted cells, we saw that the hiNPCs did not become astrocytes or formed a glial scar, which usually impacts the cell transplantation negatively as it may inhibit neuronal differentiation and therefore halt functional recovery. Rather, the hiNPCs differentiated into neurons and some even to motor neurons, characterized by expression of choline acetyl-transferase (ChAT) and motor neuron and pancreas homeobox 1 (HBP/MNX1). Performing a more thorough cell faith study to understand the broad range of cell types hiNPCs treated with EI-tPa could give rise to, perhaps using single-cell RNA sequencing, will be conducted in the future, especially when going from bench to bedside.

This was an unexpected turn of events. EI-tPa treatment improved the survival and rate of differentiation of hiNPCs. In addition, we observed a significant increase in the number of axons extending caudal to the injury site in the El-tPa-treated group accompanied by an increased locomotor capacity.

Our studies showed that the right environment is key for the stem cell transplantations to work. Although stem cell technologies provide an unprecedented potential in treating and curing previously incurable conditions, if they are not supported with the right environment and factors, they might not be able to operate under optimal conditions or could lead to worsening of the conditions.

Future studies should focus on understanding the mechanisms by which EI-tPa enabled the differentiation of NPCs and survival in the site of injury. This would greatly improve stem cell therapy, especially in neurological disorders where accessing the tissue is very challenging. Efforts should also focus on developing new or improving existing scaffolds, similar to EI-tPa, that could complement future stem cell therapy for neurodegenerative disorders.

Our study is a great example of the use of El-tPa as a scaffold, which improved not only the stem cell transplantation but also improved survival, rate of differentiation of human NPCs into functional neurons, which resulted in improved motor recovery of the rats after SCI and did not worsened pain. It also changed the expression of pain related genes at 4 months, possibly indicating a potential sensory function recovery in the long term. Stem cell therapy in combination with scaffolding molecules has a great potential of yielding to efficient treatments, which patients with a wide range of neurological disorders could benefit from.

Read Pinar’s recent article, “Tissue-type plasminogen activator-primed human iPSC-derived neural progenitor cells promote motor recovery after severe spinal cord injury”, here>>