MSc in Molecular Medicine Module

MSc in Molecular Medicine
Module: MM309
Stem cell Technology and Therapeutics
Journaling Assignment 2
Stem cell therapy for Amyotrophic lateral sclerosis
Submitted to Dr. Tan Eng Lai
Date: 14/10/2018
By
Rajesh Vivekanandan
Student ID: 00000028782
Introduction
Amyotrophic lateral sclerosis (ALS) is one of the neurodegenerative disorders causing progressive damage of both upper and lower motor neurons in the brainstem and spinal cord leading to denervation of the lateral column (lateral sclerosis) and atrophy of the muscles (amyotrophy).

The incidence rate of ALS is 1-3 per 100,000 people in North America and Europe. ALS is mostly sporadic and less familial (10%) and inherited as an autosomal dominant trait. Mutations in C9orf72(open reading frame 72 on chromosome 9) accounts for 45-50% of familial and 5% of sporadic cases and another 20% of familial ALS patients have mutations on cytosolic enzyme SOD1 (superoxide dismutase).
Exact aetiology of ALS is still unknown, several pathophysiology mechanisms like neuroinflammation, excitotoxicity, oxidative stress

Progression of ALS is persistent leading to death due to respiratory paralysis. Median survival is 3-5 years.

No treatment is currently available to stop the disease progression. Drug Riluzole, approved by FDA has minimal effect in prolonging the survival for 3 months. Another drug Edaravone, (FDA approved) has a slight improvement in the ALS disability scale.
Several types of stem cell lines used for the treatment of ALS mouse model and have shown positive effects in most of the studies. In most animal studies, transgenic animals with SOD1/G93A (SOD1 gene with Gly93Ala mutation) which is present in both familial and sporadic patients with ALS.

Research Article -1
Intravenous Mesenchymal Stem Cells (MSCs)Improve Survival and Motor Function in Experimental Amyotrophic Lateral Sclerosis ADDIN RW.CITE{{12 Uccelli,Antonio 2012}}(1)
Aims
To find the effects on survival and motor abilities in symptomatic ALS mouse model with SOD1/G93A mutation through Intravenous injection of Mesenchymal stem cells (MSCs).

To detect the effects of MSCs on histopathological features, oxidative stress and 3H D-aspartate release in SOD1/G93A mice.

Methodology
The sample of 92 transgenic mice was divided into 3 groups with an equal sex ratio
Saline-treated transgenic non-mutated SOD1
Saline-treated transgenic mutated SOD1/G93A
MSCs treated transgenic mutated SOD1/G93A
Bone marrow-derived MSCs isolated from 6-8 weeks old female C57B1/6J mice were used for this research and a dose of 1×106 cells of MSCs were injected intravenously in the test group and phosphate buffered saline (PBS) was injected in the control group.

Observational data about lifespan and motor abilities were recorded accordingly. Serial sections of the brain and spinal cord of all the experimental mouse were made and stained for histological and immunofluorescence. Genetic expression tests were done by RT-PCR.

All the data obtained were expressed as mean ± SEM (standard error of the mean), and the significance of p-value was set to <0.05.
Survival time was depicted by Kaplan-Meier plot.

Mean values of all groups were analysed by Mann-Whitney test.

Comparison of means was done by ANOVA followed by Bonferroni test (INSTAT software).

Merits
This research was done based on previous studies on bone marrow-derived MSCs on the pre-symptomatic phase of transgenic ALS mice models which provided significant improvement of survival and motor abilities. The results showed that MSCs were able to extend the survival and motor performance even after the disease process has occurred, which proves the research hypothesis.

The results suggest that MSCs when injected through the intravenous route, can influence the CNS, but the concentration of the cells decreases eventually. Hence there will be a need for repeated injections to provide a long-term benefit in human ALS patients.

The study provides statistical evidence which showed that MSCs even when injected through the systemic route can provide neuroprotective effect and enhanced antioxidative property which helps to delay the disease progression of ALS.

Demerits
The number of animals used in each group was not mentioned clearly in the study design.
The study was limited only to the intravenous administration of MSCs in the symptomatic ALS transgenic SOD1/G93A mice. Comparison of other routes of administration like the intraspinal and intrathecal route where the MSCs are directly transplanted into the CNS was not considered.

The neuronal differentiation of the MSCs after the injection was not evaluated.

Conclusion
The study fulfilled the proposed hypotheses and encourages further evaluation of BM-MSCs on human trails because of their safety profile, which was proved by other clinical studies for various diseases. Overall the study supports the usage of BM-MSCs for the treatment for ALS.

Research Article -2
Transplantation of Human Adipose Tissue-Derived Stem Cells Delays Clinical Onset and Prolongs Life Span in ALS Mouse ModelADDIN RW.CITE{{14 Kim,KwangS. 2014}}(2)
Aims
To identify the effects of human adipose tissue-derived stem cells (ASCs) on disease onset and lifespan in ALS mouse model with SOD1/G93A mutation.

To estimate the neuroprotective effects of the growth factors secreted by ASCs.

Methodology
In this study, adipose tissue-derived stem cells were isolated from 1 female and 2 male donors through liposuction and informed consent was taken for research use and the study was approved by the Ethics committee.

Female transgenic mice with SOD1/G93A mutation were selected for the study and were divided into three groups with 9 mice in each group
Intravenous (IV) group (received 1×106 cells of ASCs)
Intracerebroventricular (ICV) group (received 2×105 cells of ASCs in 3 different zones
The control group (received phosphate-buffered saline)
Observational data about the onset of disease and lifespan. Spinal cord sections were stained for immunohistochemistry. Gene expression studies were done by RT-PCR. Cytokines concentration was measured by ELISA kits for multiple growth factors. TUNEL staining assay was performed to identify apoptotic cell death.
Data points were expressed as mean ± SEM and one-way ANOVA (with p<0.05) was used to determine the significance among the tested groups. Survival data was determined by the Student-Newman-Keuls test and analysed by Kaplan-Meier curve. SPSS software was used to test the significance.

Merits
The study uses human adipose tissue-derived stem cells (ASCs) which are considered as an easy alternative to BM-MSCs. Transgenic SOD1/G93A mice treated with ASCs through the ICV route showed a significant delay in the onset of disease and extended the lifespan compared to other groups. ASCs administered through IV route had minimal effect on survival and disease onset.

The study showed ASCs transplanted through the ICV route did not differentiate into neural cells but secreted various growth factors (maximum effect by VEGF & IGF1) which has neuroprotective effects that block neuronal cell death resulting in slow progression of the disease process in transgenic ALS mice.

Demerits
The sample size of the study was very minimum and hence the results can be misleading.

Only female transgenic mice models were selected in the study design which results in gender bias.

The percentage of ASCs survived after the ICV transplantation procedure was very minimal.

The effect of ASCs on the common pathogenic processes of ALS (excitotoxicity, neuroinflammation, oxidative stress) was not studied.
Conclusion
The study suggests that ASCs transplanted ICV route can extend lifespan and disease onset, but the statistical data seems to be limited. Hence the significance of the findings cannot be extended to human clinical trials. This preliminary study done with better study design can create a good impact in the field of stem cell therapy for ALS.

Research Article -3
Focal Transplantation of Human iPSC-Derived Glial-Rich Neural Progenitors Improves Lifespan of ALS MiceADDIN RW.CITE{{9 Kondo,T. 2014}}(3)
Aims
To see the effects of hiPSC derived GRNPs on lifespan and motor abilities in diseased ALS mouse model with transgenic SOD1/G93A mutation.

To find the differentiation and tumorigenic potential of the transplanted hiPSC-GRNPs through intraspinal administration in transgenic mice model.

Methodology
A Sample set of 24 transgenic mice with 17 males and 7 females were taken as the test group and another set of 24 transgenic mice with a sex ratio of 17:7 (male: female) were taken as control group. This study was approved by CiRA animal experiment committee.

40,000 hiPSC-GRNPs were transplanted into each side of ventral horns of the lumbar spinal cord of the transgenic SOD1/G93A mice through intraspinal injection on the 90th day of life. Clear PBS solution was injected into mice of the control group through the same approach.

Histological analysis of the spinal cords near the injection site was performed to study for engraftment and immunostaining was done to group the cell subtypes near the graft site. Quantitative RT-PCR and Western-Blot analysis were used to find the gene expressions.
Survival data from the two groups were evaluated using the Kaplan-Meier plot and log-rank test was used to compare the curves. Two-way ANOVA followed by Tukey-Kramer test was used for analysis of motor function. Mann-Whitney test with the p-value <0.05 significance limit was used for the mean analysis from the two groups.

Merits
The study was designed in a perfect way with an equal number of cases and control with the same-sex ratio in both groups. The methods used in the study and the representation of the statistical data were clear to ensure the research’s repeatability.
The animals transplanted with GRNPs showed improved lifespan than the control group and this result was maximum in the male transgenic SOD1/G93A mice treated with hiPSC-GRNPs. This proves that GRNP has a positive effect in improving the survival time of the diseased ALS mice.

Transplanted GRNPs were derived from human iPSC cell line 201B7 clone which showed low tumorigenicity. Post-transplantation studies in the test group confirm the differentiation of GRNPs into mature astrocytes with no new tumour formation at the injection site or in any other organs.
Demerits
The study was confined only to the extension of lifespan. Improvement in motor abilities was not evaluated, which is also an important aspect in the treatment of ALS affected patient.

The study cannot be extended to clinical human trials because the long-term effects of transplanted GRNPs were not studied and risk for delayed cancer formation is anticipated.

Conclusion
The study was planned effectively, and the results obtained proved the hypothesis that hiPSC-derived GRNPs help in prolongation of the lifespan of the diseased transgenic SOD1/G93A mice. This research opens the way to study other effects of human iPSCs-derived GRNPs when injected into the spinal cord at various levels. Overall the study suggests the use of Human iPSCs for the treatment for ALS after further evaluation.

Research Article -4
Transplantation of Mesenchymal Stromal Cells in Patients with Amyotrophic Lateral Sclerosis: Results of Phase I/IIa Clinical TrialADDIN RW.CITE{{21 Syková,Eva 2017}}(4)
Aims
To determine the safety and clinical efficacy of autologous BM-MSCs transplantation in patients with ALS.

Methodology
The study design was prospective, non-randomized, open-label, without a placebo control group and single centered. Informed consent was obtained by all the participants of the trial and the study was approved by the Ethics Committee.

26 patients were selected for the study following a standardised inclusion and exclusion criteria. The patients were subjected to 6 months of pre-transplantation screening and 18 months of post-transplantation check-ups at regular intervals.

12 ml of Bone marrow were aspirated and expanded under GMP conditions into vials containing 15 ± 4.5 x 106 cells of MSCs. Each patient received a single injection of this vial through the intrathecal route.

All the patients were hospitalized up to 3 days after the transplantation to screen for any immediate adverse reactions and all the necessary clinical investigations were recorded.

ALS functional rating scale (ALSFRS), Forced vital capacity (FVC) and muscle weakness scale (WS) were used for the assessment of the efficacy.

Data from all the patients were collected at regular intervals during both pre and post-transplantation period and they were compared. Regression analysis method was used for evaluating the efficacy and paired t-test was used for comparing the slopes for correlated variables.
Merits
The study proved that autologous MSCs were safe to use in human trials as there were no severe adverse reactions recorded in any of patients who received MSCs.
Direct transplantation of MSCs into the CNS via intrathecal route was proven effective in animal models and previous clinical trials. This study enhances the safety profile of the Intrathecal route for administration of MSCs.

Efficacy of the MSCs was evaluated among 23 patients in this study and results proved limited but significant slowdown in the disease progression. This result correlates with the earlier studies on MSCs which reduced the ALS disease progression by secreting neuroprotective growth factors (VEGF, BDNF ; ?NGF).
Demerits
The selection criteria of the patient did not include the onset of the disease (sporadic or familial).

The check-up intervals were longer, the results obtained possibly affected by the recall bias of the patients.
The requirement of the second dose for prolonging the therapeutic effect of MSCs was not evaluated.
Conclusion
This clinical trial clarifies that autologous MSCs introduced through intrathecal route are safe for treatment in human subjects. The MSCs are proved to be effective in decelerating the disease progression maximum at the first 6 months and hence they are to be injected at regular intervals for maximum benefit.
Conclusion
The stem cell therapy for Amyotrophic lateral sclerosis (ALS) has shown positive results on the animal and human trials using various stem cell types. The stem cells (MSCs, ASCs ; hiPSCs-GNRF) aided in preventing the neural cell death
by promoting secretion of the neuroprotective growth factors (like VEGF, IGF1, BDNF ; ?NGF)
decreasing the neuroinflammation by inhibiting the activation of astrocytes and microglial cells
inhibiting the oxidative stress and
downregulation of the glutamate toxicity.

The mode of introduction of the stem cells into the host is a crucial factor in the treatment for ALS because many cells introduced through the peripheral approach (IV) did not enter the CNS when compared to the direct transplantation into the CNS (intrathecal).

For finding the suitable cell for long-term treatment in ALS, researchers are using various cell lines like MSCs, WJ-MSCs, NSCs, MSCs-GDNF. Similarly, for the introduction of stem cells into the patient scientists are using nanocarrier technology.
Stem cell research for ALS is happening for more than 20 years and recently scientists have proved that stem cell therapy is safe for human usage. Hence there is a need for further evaluation of efficacy involving many study subjects, more comprehensive multi-center study with expert scientists and strong study design in the future.

Definitive treatment for Amyotrophic lateral sclerosis is possible through the stem cell research.
References
ADDIN RW.BIB(1) Uccelli A, Milanese M, Principato MC, Morando S, Bonifacino T, Vergani L, et al. Intravenous Mesenchymal Stem Cells Improve Survival and Motor Function in Experimental Amyotrophic Lateral Sclerosis. Molecular Medicine 2012 03/30;18(1):794-804.

(2) Kim KS, Lee HJ, An J, Kim YB, Ra JC, Lim I, et al. Transplantation of human adipose tissue-derived stem cells delays clinical onset and prolongs life span in ALS mouse model. Cell Transplant 2014;23(12):1585—1597.

(3) Kondo T, Funayama M, Tsukita K, Hotta A, Yasuda A, Nori S, et al. Focal transplantation of human iPSC-derived glial-rich neural progenitors improves lifespan of ALS mice. Stem Cell Rep 2014;3(2):242-249.

(4) Syková E, Rychmach P, Drahorádová I, Konrádová Š, R?ži?ková K, Vo?íšek I, et al. Transplantation of Mesenchymal Stromal Cells in Patients with Amyotrophic Lateral Sclerosis: Results of Phase I/IIa Clinical Trial. Cell Transplant 2017 04/01; 2018/10;26(4):647-658.