Werdnig Hoffman is an untreatable neuromuscular disorder caused by reduced expression of the SMN protein, leading to the loss of MNs in the spinal cord. Conducting clinical trials in WH patients is extremely difficult due to the medical fragility of these patients and the frequent development of respiratory illnesses among these children. Stem cell therapy is a potential therapeutic approach for spinal muscular atrophies, as it results in the activation of molecular and cellular mechanisms that support endogenous neuronal activities and protects against neurodegeneration.
As with all clinical trials in the first phase, what is important is the safety of the intervention. Our study showed that allogeneic side population adipose-derived mesenchymal stem cells in patients with Werdnig Hoffman is safe. In this clinical trial, the cell expansion process did not involve any alteration to the genome of the cells in any of the cases. In 2015 Villanova and Bach reported the safety of allogeneic mesenchymal stem cells from different sources and protocols in three patients with WH . Selection of the proper cell type and administration of the appropriate amounts of cells is very important. The cells must exhibit the characteristics of neuroprotection while avoiding the potential for tumor formation . Also, issues regarding cell graft survival must be examined within the transplanted microenvironments, as well as immune rejection potential. Many studies show us that while MSCs move into areas of damage, they only survive for a short period and cannot be found in the CNS parenchyma a few weeks after administration . However, when MSCs directly injected into the sciatic nerve following a crush injury prevented denervation of neuromuscular junctions and improved motor performance .
Recent reports using bone marrow stem cells in the clinical trial of ALS has shown preliminary hopeful results in the motor performance of transplanted patients [43–45]. This indicates that trophic factors could recover and stimulate the function of MNs, or rescue neurons in reversible phases of cell death. Surely we had no expectation of the differentiation of SPADMSCs into motoneurons. The confined differentiation of stem cells in the spinal cord has been previously reported using neural stem cells [46, 47], while, these stem cells were able to differentiate in the brain [48, 49]. Probably the spinal cord has not an inductive capacity to develop a neural phenotype from undifferentiated stem cells. However, It should be noted that the beneficial effects due to neurotrophic factors were first described in the 80's .
For the first time, stem cells transplantation was used in a SMA model as a putative therapeutic pathway in 2008 . In this clinical trial, we have chosen a population of the MSCs because of their immunomodulatory capacity to neutralize neurotoxins and exert their neuroprotective potential by producing bioactive neurotrophins. Also, we counted on the possibility of stimulating local progenitor cells to replace SMN1 protein. Motor neurons are unlikely to be the only cellular population of the nervous system that is affected in SMA. Disrupted sensory pathways have been described in severe SMA patients . Sometimes the disruption of functional connectivity between sensory and motor neurons in the spinal cord occurs that can worsen the patients' disease phenotype. So not only protecting the motoneurons but also preventing the destruction of these sensory neurons in the spinal cord may play an important role in improving the phenotype of WH patients.
In 2012, Carrozzi et al., reported no benefits from bone marrow MSC infusions for five WH patients . They have reported the outcome in less than one month of cell administration and subsequently other investigators have denied the validity of earlier positive outcomes of cell therapy for WH patients on the basis of Carrozzi report [53–55].
Based on the frustrating results obtained so far, WH patients currently have little choice for effective treatments, which is restricted to expensive gene therapy, so clinicians have been limited to only treat the secondary complications arising from the disease especially pulmonary complications that lead the patients to death. This study on five patients with WH shows that transplantation of SPADMSCs into the lumbar spinal cord appears to be a safe procedure that causes no major, short- or medium-term, deleterious effects. No patients suffered side effects from SPADMSC treatment except for mild fever in the two patients under the intervention. All patients in the control group died within an average of nine months (Fig. 5A), but in the cell therapy group, one patient is alive after follow-up for up to 24 months and he has improved chest muscle movement. This patient, who was also weightier than the other patients, received the highest SPADMSCs dose (on his weight) than others (Fig. 4B). Since the adipose tissue donors in this project were all female, therefore, the donor gender cannot be considered as one of the influencing factors in the differences between mesenchymal stem cell function.
Our findings emerge from the similar clinical protocols that are comparable to these studies, but we, in turn, employed a more potent side population cells and much higher cell dosage. The first founded result is that the CSF cell injection in human appears to be a safe, reproducible and reliable procedure. Also, the perspective improvements were made here, particularly concerning the number of administered cells, that in our study was five times higher than in previous approaches – a maximum of 5 × 106 cells/kg used here versus the earlier 1 × 106 cells . It is possible to administer this amount of cells in a volume of only 3 ml using SPADMSCs whereas, it is impossible to use this dose of MSCs if it was prepared by the conventional methods.
Careful attention must be paid to the technical aspects of cell delivery within the nervous system. As MSCs could pass the blood-brain barrier we did chose intrathecal delivery of the cells to get the most effect of injected cells on the desired site. In many studies, the stem cells were injected intravenously or in combination with intrathecal route . In the intravenous (IV) delivery route MSCs mostly distribute in the lungs, and then in the spleen, liver, bone marrow, thymus and kidney they will spread . Other studies showed that the total body count of labeled MSCs by bioluminescence method revealed a decrease from the intensity measured at fifteen minutes post IV infusion to about 60% after 24 hours and less than 10% after 72 hours .
Electrophysiological outcomes include the estimation of the numbers of motor units and motor action potential amplitude. The increase in motor action potential amplitude captures both an increase in the number of motor units and the presence of collateral sprouting of axons from adjacent surviving motor neurons . In WH patients spontaneous rhythmic firing of motor units have been recorded and it shows some short and low amplitude potentials in addition to long duration, high amplitude potentials . In our study, there were no significant differences in the amplitude of motor responses in any of the upper and lower extremity nerves between the two groups after the first injection. We found that in the tibial nerve, the mean of the amplitude response of the motor in the cell therapy group was 0.54 mV with a significant difference to the control group after the third injection (Table). This may have occurred through the release of neurotrophic and growth factors, cytokines and immunomodulatory molecules that diffuse into the pathological tissue, thereby eliciting neuroprotective effects. Also, there are pieces of evidence now exist that neural stem cells, which integrate and survive in the brain parenchyma, exert their immunomodulation ability through secretion of extracellular membrane vesicles or exosomes, influencing the microenvironment through the traffic of bioactive molecules [60, 61]. Xin and colleagues showed that intravenous injection of MSC-derived exosomes induces neurogenesis, neuronal repair, and angiogenesis after stroke . In addition, mesenchymal stem cell-derived exosomes induce axonal growth through the transfer of microRNAs to the neuronal cell body, which may be a novel approach in the treatment of progressive neurodegenerative diseases .
We have been able to reproducibly expand SPADMSCs ex-vivo. Methodological varieties in the isolation, processing, delivery, and the assessments between those referred to in the letter and those in our trial could have played an important role in the difference in patients' treatment outcome. This trial is actually based on the use of only three stem cell donors. The use of such a limited number of donors inherently reduces the inter-treatment variability that may arise from the implantation of different stem cell lines into different patients.
Very few studies in the scientific literature report the results of clinical trials with stem cell transplantation for Werdnig-Hoffman patients. These results are not definitive and no trial has been replicated in multiple centers. Therefore, we believe that instead of preventing these kinds of clinical trials, it is better to learn from the previous trials, develop more efficient methods of safe cell therapy for these patients and to investigate them as a multi-center study.