Investigating the Effects of Immunosuppressants on Neural Stem Cell Therapy

1. Abstract

 

​Alzheimer's disease afflicts more than 55 million people around the world, leading to memory loss, impaired cognitive abilities, and difficulties in performing daily tasks. First identified by Alois Alzheimer in 1906, the disease results from neuron loss in various brain regions. As the most prevalent form of dementia, Alzheimer's poses a significant healthcare challenge, with no current cure. While existing treatments offer only symptomatic relief without altering the disease's progression, ongoing research is exploring innovative approaches, including the use of Neural Stem Cells (NSCs) and other immunomodulatory drugs, to achieve lasting results.

The primary question this project seeks to address is how to enhance the effectiveness of NSC therapy for Alzheimer's disease using Immunosuppressants. A significant challenge with NSC therapy is the limited retention of neural stem cells over time due to immune responses and other factors. Research indicates that immunodeficient SCID mice, which have a compromised immune system, exhibit reduced neuronal damage and improved survival of neural stem/progenitor cells following a stroke compared to immunocompetent wild-type
mice. (Saino et al., 2010)

Based on this insight, I hypothesized that combining NSC therapy with immunosuppressants could mitigate the immune response against NSCs, prolonging their retention and enhancing their therapeutic efficacy. Preliminary studies suggest that immunosuppressants such as Tacrolimus (FK506) may offer a promising approach to address
this challenge.

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2. Introduction

 

a. Alzheimer's Disease: A Brief Overview

Alzheimer's disrupts vital neuronal processes, including communication, metabolism, and repair. Key pathological hallmarks of Alzheimer's include the accumulation of amyloid beta (Aβ) plaques and neurofibrillary tangles in the brain. Aβ is formed from the breakdown of a larger protein, the amyloid precursor, while neurofibrillary tangles are abnormal accumulations of tau protein within neurons. These pathological changes lead to the disruption of synaptic communication between neurons, contributing to the cognitive decline observed in Alzheimer's patients. In healthy aging, the brain shrinks slightly without significant neuron loss. However, in Alzheimer's, neurons malfunction, lose connections, and eventually die, disrupting vital brain processes like communication, metabolism, and repair. Alzheimer's disease progresses through three distinct stages: Pre-clinical, Pro-Dermal, and Severe Dementia. In the Pre-clinical stage, individuals may not display noticeable symptoms, but subtle changes in cognitive function, such as the development of amyloid-beta plaques and tau tangles, begin to emerge. Biomarker evidence of amyloidosis can be detected through PET imaging or cerebrospinal fluid (CSF) analysis during this stage. As the disease advances to the Pro-Dermal stage, symptoms become more pronounced and can include increased memory loss, confusion, difficulty with language, and challenges with problem-solving. During this phase, Alzheimer's disease predominantly affects the cerebral cortex of the brain. In the final stage, Severe Dementia, individuals experience significant impairment in social and occupational functioning, marked by severe cognitive deficits that affect daily activities. Understanding these stages is essential for early diagnosis, intervention, and appropriate management of Alzheimer's disease.

 

b. Current Treatment Landscape

Currently, available treatments for Alzheimer's are primarily symptomatic, offering temporary relief without modifying the disease's course. Cholinesterase inhibitors such as Galantamine, rivastigmine, and donepezil are prescribed for mild to moderate Alzheimer's symptoms. Recently, Lecanemab, an FDA-approved immunotherapy targeting beta-amyloid, has shown promise in reducing amyloid plaques. However, its use is associated with potential side effects, including brain swelling, bleeding, and gastrointestinal issues.

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c. The Promise of Neural Stem Cell (NSC) Therapy

Emerging research has highlighted the potential of Neural Stem Cell (NSC) therapy as a promising alternative to AD treatment. NSCs are self-renewing, multipotent cells capable of generating all major cell types in the adult central nervous system. They offer a unique opportunity to regenerate lost neurons and establish synaptic connections. Recent studies have shown that NSCs, upon transplantation, not only replace damaged cells and modulate inflammation, promote healing, and communicate with the host's cells, leading to positive effects on brain cells. 

In the context of Alzheimer's, NSC therapy targets the regeneration of degraded cholinergic neurons and the establishment of synaptic connections with neighbouring neurons. However, challenges persist, such as determining the optimal timing for transplantation relative to disease progression and ensuring the long-term efficacy of NSC-based treatments. One major obstacle to successful stem cell transplantation in Alzheimer's treatment is the brain's natural immune response, where CD4+ and CD3+ T cells attack transplanted cells, reducing their retention. Research suggests that immunosuppression can enhance the migration of neural stem cells to the injury site and promote tissue regeneration (Chen et al., 2023). To address this, combining NSC therapy with immunosuppressants like Tacrolimus (FK506) and CD4 antibody can effectively mitigate immune responses and enhance stem cell retention. 

Moreover, the accumulation of chronic microglia and glial cells, along with increased astrocytes, exacerbates brain inflammation, compromising NSC survival and further
damaging the neurons they are intended to support and protect (NIH, 2024). By modulating these inflammatory responses, immunosuppressive strategies can not only improve NSC survival but also enhance their therapeutic potential in Alzheimer's disease 

 

d. Immunosuppressants and Stem Cell Therapy 

After reviewing various studies on immunosuppressants and their impact on the brain, including drugs like Glatiramer Acetate (Copaxone), Rapamycin, Thalidomide & Derivatives, Minocycline, Cyclosporine, and Tacrolimus (Munafò et al., 2020), and assessed their effects on cognitive improvement in Alzheimer's patients and levels of Aβ, p-tau, and neurodegeneration. Based on these findings, I determined that Tacrolimus (FK506) is most effective in mitigating negative immune responses in stem cell therapy (McGinley et al., 2017). Additionally, combining it with a CD4+ antibody can further reduce inflammation by blocking T-cell receptors, addressing another significant factor contributing to increased inflammation. 

I aim to bridge the research gap by integrating two separate treatments, hypothesizing that they can mutually address each other's challenges. To test this, I devised a hypothetical experiment and conducted statistical and analytical tests based on prior research and calculations. My research focuses on enhancing the effectiveness and retention of NSC therapy in Alzheimer's models. I aim to identify the optimal combination of immunosuppressants, considering type, dosage, and location, to maximize therapeutic outcomes using human NSCs (hNSCs) and human induced pluripotent stem cell-derived neural progenitor cells (hiNPCs) in various mouse models.

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3. Materials

 

a. Animal Models

• APP/PS1 rat: Represents the pro-dermal stage of Alzheimer's Disease

• 3xtg-AD rat: Represents the pre-clinical stage of Alzheimer's Disease

• LE rat (Control variable) 

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b. Stem Cells

• hNSC: HSI-HK532-IGF-1 

• hiNPC 

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c. Immunosuppressants

• Tacrolimus (FK506) + CD4 antibody in three different doses 

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4. Methods

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a. Type of Rat Models:

The selection of rat models for this experiment was purposefully made based on specific criteria. APP/PS1 rats are genetically engineered to express mutant forms of human amyloid precursor protein (APP) and presenilin 1 (PS1). The mutation in APP leads to an increased production of amyloid-beta (Aβ) peptides, which are known to aggregate and form plaques in the brains of Alzheimer's patients. The PS1 mutation affects the processing of APP, further increasing Aβ production. The APP/PS1 rat model is used to study the accumulation of Aβ plaques and the progression of Alzheimer's disease (Agca C et al.) The 3xTg-AD rat model carries three genetic mutations associated with Alzheimer's disease: APP, presenilin 1 (PS1), and tau. Similar to the APP/PS1 model, the mutations in APP and PS1 result in increased Aβ production and plaque formation. The inclusion of the tau mutation allows researchers to study the development of neurofibrillary tangles, another hallmark pathology of Alzheimer's disease. This model provides a more comprehensive representation of the disease pathology seen in humans (Javonillo et al., 2022).

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b. Type of NSCs:

hNSC (Human Neural Stem Cells): Originating from human neural tissue, hNSCs can differentiate into neurons, astrocytes, and oligodendrocytes. The "HSI-HK532-IGF-1" subtype may feature specific modifications like IGF-1 overexpression. I selected this line of hNSCs because of multiple positive studies that had used this line of Stem cells (McGinley et al., 2017). 

hiNPC (Human Induced Pluripotent Stem Cell-derived Neural Progenitor Cells): Derived from reprogrammed human-induced pluripotent stem cells (hiPSCs), hiNPCs offer greater versatility in neural cell differentiation due to their pluripotent origin (Shiga et al., 2019). 

In summary, while hNSCs are sourced directly from neural tissue, hiNPCs are derived from hiPSCs, making them more adaptable and versatile in terms of differentiation potential. This makes it a crucial element to this experiment allowing us to as close as possible how this would be in real life. 

 

c. Experimental Procedure:

The experiment will be separated into 2 sections. The first will use hNSC, HSI-HK532-IGF-1, injected into 18 rats: 6 for each disease stage + the control. For each stage, three doses of Tacrolimus and CD4 antibody will be administered daily for 14 days into two different brain areas: the hippocampus and the corpus callosum. This process will be repeated using hiNPC.

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d. Data Collection and Analysis:

The experiment will assess stem cell longevity at 4, 8, and 16 weeks post-injection through a cell count and will be analyzed by an ANOVA test. Additionally, it will analyze NSC proliferation, and the differentiation into mature neurons, through the cell’s action potential (AP). Fluorescence intensity tests that will detect KCC2 and Calcium-binding protein (CaBP) biomarkers can tell us if the stem cells have become mature. Additionally, An Immunohistochemistry test (IHC) will monitor microglial activity and potential changes in Aβ and tau proteins related to Alzheimer's cognitive improvement. The experiment will also administer two cognitive memory tests, the Morris water maze (MWM) for spatial memory and the novel object recognition (NOR) test for memory retention. In the MWM, animals are placed in a large pool of opaque water where they must locate a hidden platform submerged just beneath the water's surface. The animals use spatial cues around the room to navigate and find the platform. Over multiple trials, the time taken by the animal to find the platform (latency) is recorded. A shorter latency over subsequent trials indicates improved spatial learning and memory (Vorhees & Williams, 2006). In a NOR test, the animal is initially placed in an open arena containing two identical objects, which the animal is allowed to explore freely. After a certain period, one of the familiar objects is replaced with a novel (new) object. The animal is then reintroduced to the arena, and its preference for exploring the novel object over the familiar one is measured. The underlying principle is that animals have a natural tendency to explore new or unfamiliar objects more than familiar ones. Thus, increased exploration time of the novel object compared to the familiar one is considered an indicator of intact memory retention and recognition (Ennaceur & Souza, 2018). Additionally, an observation chart will document post-transplantation factors to enhance understanding of the experimental outcomes. 

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5. Predicted Results

The predictions are formulated based on prior experiments and are theoretically grounded. The statistical tests utilized calculations derived from averaging previous studies or making educated estimates to provide the most probable outcomes. For instance, insights from studies comparing the effects of NSCs and NPCs, or evaluating the influence of varying immunosuppressant doses, were used to inform my hypotheses and anticipate the likely results of this experiment.​

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​Figure 1.1​

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1.1 - ANOVA test indicating the length of duration each human NeuroStemcell stayed 
in the brain. It measures the retention time of the cells, considering variables like injection site and Tacrolimus (FK506) + CD4 antibody dosage. The figure represents the Mean with the Standard deviation. Figures were made using GraphPad. 

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Figure 1.2

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Figure 1.2 - ANOVA test indicating the length of duration each human-induced NeuroPrognator cell stayed in the brain. It measures the retention time of the cells, considering variables like injection site and Tacrolimus (FK506) + CD4 antibody dosage. The figure represents the Mean with the Standard deviation. Figures were made using
GraphPad.

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Figure 1.3

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​Figure 1.3 presents the ANOVA test analysis conducted on hNSCs within the brain, which allowed me to create the graphs. 

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Figure 2

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Zhang et al., (2019) [Figure 1.4] 

Figure 2 - illustrates the Action Potential (AP) of hiNPCs in the brain post-transplantation into the hippocampus. While this assessment was conducted without the use of immunosuppressants, it serves as a hypothetical representation. It's anticipated that with immunosuppressants, the results could potentially be as depicted or even more favourable.

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Figure 3

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Fortes de Valencia et al., (2003). [Figure 3] 

Figure 3 - An example of a Fluorescence intensity test from a research lab at the University of Sao Paulo.

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Figure 4.1 & 4.2

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McGinley et al., (2018) [Figure 4.1 & 4.2]
Figure 4.1 & 4.2 - These Immunohistochemistry (IHC) images show the presence of Aβ plaques in the hippocampus and cortex of the APP/PS1 (Pro-Dermal) mouse brains. The sham group (not treated with NSCs) shows significant plaque formation. In contrast, the NSC-treated mice display reduced plaque levels. (McGinley et al., 2018)

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Figure 5.1 & 5.2

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Figure 5.1 & 5.2 - ANOVA test indicating the amount of time each group took to explore the novel and familiar object after 8 weeks and 16 weeks of transplantation. The figure represents the Mean with the Standard deviation. Both figures were made using GraphPad. 

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Figure 5.3

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Figure 5.3 - The ANOVA analysis of raw data for the NOR assessments.

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Figure 6

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Chen et al., (2015)[ Figure 6] 

Figure 6 - This is an example of a MWM test done on a study in 2015. The test used NSC transplantation on an AD model and presented representative movement traces from all four groups on Day 4 of the navigation session. However, this study did not use Immunosuppressants (Chen et al., 2015).

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Figure 7

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​Figure 7 - This observation chart was made by correlating results from different studies. (Chen et al., 2023) (McGinley et al., 2018)(Hayashi et al., 2020)(McGinley et al., 2017)

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6. Discussion

The ANOVA analysis, as shown in Figures 1.1 and 1.2, assesses the retention duration of hNSCs and hiNPCs in the brain, taking into account variables such as injection site and Tacrolimus (FK506) + CD4 antibody dosage. To hypothesize the data results, I first calculated the success rate for each dosage within each model. This analysis involved reviewing past experiments and their outcomes and taking into account both the mean and mode. In instances where limited information was available for certain models, I conducted an educated analysis to make informed predictions. Next, I needed to determine the rate of decrease, which required a more theoretical approach. By considering factors such as the success rate of NSC cells across various models and other individual variables, and averaging these values with data from previous studies, I developed a unique calculation for each NSC group.

 

The outcome of this highlights that both hNSCs and hiNPCs perform optimally with a 3 mg/kg dosage of FK506 and CD4 antibodies at the Corpus Callosum in the pro-dermal model. This dosage consistently surpassed the 0.3 mg/kg and 1.5 mg/kg options across all models. Supporting this, a 2017 study indicated that the 1.5 mg/kg dosage is near the immune rejection threshold for transplanted NSCs, even when using Tacrolimus (McGinley et al., 2017). In essence, the 3 mg/kg dosage yields the best results for both cell types. While hNSCs showed inconsistent success rates, hiNPCs consistently outperformed them, suggesting hiNPCs may be a more stable treatment option. The most promising outcomes were observed with the Pro-dermal model using injections at the Corpus Callosum and the Pre-clinical model with injections at the Hippocampus. This suggests that the location of the injection may be crucial at different stages of Alzheimer's Disease (AD). AD commonly begins in the hippocampus. Therefore, during the Pre-clinical stage, injecting stem cells into the Hippocampus may be most beneficial. As the disease progresses and affects the Cerebral Cortex, which is near the Corpus Callosum, therefore, injections at the Corpus Callosum could be more advantageous during the Pro-dermal stage of AD

 

Figure 2 illustrates the assessment of hiNPCs in the brain following transplantation into the hippocampus. The potential of these stem cells is evaluated not only in terms of longevity but also quality. The data indicates that hiNPCs consistently generate action potentials (APs) around the 4-6 month mark, with approximately 100% of neurons at these intervals capable of generating APs. In contrast, only 50% of neurons recorded at the 2-month mark displayed repetitive firing. The frequency-current (F-I) curves for neurons at 4- and 6-month intervals show a gradual increase in AP frequency (Zhang et al., 2019). Therefore, suggests that stem cells require time for proliferation and maturation into stable neurons. This implies that longer NSC presence in the brain increases the likelihood of maturation. Although this study didn't use immunosuppressants, the addition of Tacrolimus would likely extend cell lifespan, enhancing proliferation and maturation opportunities. Cognitive tests also indicate improved outcomes with prolonged NSC presence. 

 

Figure 3 is an example of a fluorescence intensity test from a research lab at the University of Sao Paulo. Although there is no way for me to conduct a fluorescence intensity test experiment, this is an example of what it could look like. In the context of this experiment, the results would show the KCC2 and the Calcium-binding protein when the NSCs turn into mature neurons. This test allows us to verify if the stem cells have turned into mature neurons. 

 

Figure 4 shows the presence of Aβ plaques in the hippocampus and cortex of the APP/PS1 (Pro-Dermal) mouse brains. The sham group (not treated with NSCs) shows significant plaque formation. In contrast, the NSC-treated mice display reduced plaque levels. (McGinley et al., 2018).In this study, immunosuppressants were not used. Analyzing the data suggests that the combination of immunosuppressants with the CD4+ antibody likely reduces Aβ plaque buildup. If a notable change was observed with NSCs alone, adding FK506 could amplify this effect. In addition, since the data was collected over a short period due to the limited lifespan of NSCs in the brain, the addition of immunosuppressants may prolong their presence, further enhancing this positive response. 

 

Figures 5.1 and 5.2, evaluate the cognitive abilities of the Alzheimer's disease models at 8 and 16 weeks, focusing on memory retention (Ager et al., 2015). At 8 weeks, all groups

performed the task similarly. However, by 16 weeks, the control model exhibited the shortest exploration time for the novel object, indicating a failure to recognize its novelty. In contrast, both the hNSC and hiNPC groups recognized the novel object and spent significantly more time exploring it. The data for the ANOVA tests was derived from the duration of NSCs and NPCs in the brain, success rates for each dosage, rate of decrease, and observational findings from prior studies (McGinley et al., 2018). The results indicate that the prolonged presence of stem cells in the brain correlates with increased cognitive function. Conversely, the control group exhibited declining performance over time, highlighting the cognitive deterioration experienced by Alzheimer's patients without proper treatment. So based on these results, it can be said that the use of NSC with FK506 can definitely increase cognitive application. 

 

Additionally, the example of the MWM test (figure 6) further supports the theory of NSC therapy as well. The study conducted this test using NSC transplantation on an AD model and presented representative movement traces from all four groups on Day 4 of the navigation session. Both the treatment groups and control animals exhibited similar movement distances, whereas the AD model rats displayed circuitous routes before locating the platform. It's worth noting that this test did not utilize immunosuppressants. Therefore, hypothetically, if immunosuppressants were used, AD model rats might follow more stable routes, as the NSCs would likely remain stable within the brain. (Chen et al., 2015). 

 

The observation chart I created illustrates the effects of different NSC types injected into various brain regions. I chose to focus on recording data for the groups receiving a dosage of 3mg/kg, as it demonstrated the most promising results. The chart indicates a reduction in tau proteins and an increase in synaptic density across most groups. Additionally, many groups displayed a decrease in T cells, which can be advantageous for Alzheimer's disease (AD). 

 

T cells play a crucial role in immune responses. When NSCs are transplanted into the brain, T cells can initiate an immune reaction against these foreign cells, leading to rejection or diminished efficacy. By suppressing T cell activity, the chances of NSC survival and integration into the brain improve. Moreover, T cells can exacerbate brain inflammation, further accelerating neurodegeneration in AD. By reducing T cell activity, inflammation is mitigated, potentially safeguarding neurons and enhancing the therapeutic benefits of NSCs. Incorporating immunosuppressants like Tacrolimus (FK506) and CD4 antibodies, which target T cell activity, can be a valuable approach to enhance NSC therapy for Alzheimer's disease. Consequently, the CD4+ antibody's role in modulating brain inflammation addresses a previous research limitation, providing multiple therapeutic advantages. This is corroborated by the findings in the observation chart. 

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7. Conclusions and Future Prospects.​

In conclusion, this research unveils a promising strategy for Alzheimer's treatment by combining immunosuppressants with NSC therapy. Optimal results were observed with a 3mg/kg dosage of FK506 + CD4+ antibody injected at the Corpus Callosum in the Pro-dermal Alzheimer's model, enhancing the performance of both nNSCs and hiNPCs. Prolonged retention of NSCs/NPCs in the brain promotes their maturation into healthy neurons, leading to increased synaptic density, reduced amyloid beta and tau proteins, and decreased inflammation. This extended presence translates to enhanced therapeutic benefits and notable improvements. Essentially, the longer the stem cells remain in the brain, the more favourable the therapeutic outcomes. 

Moving forward, the immediate priority is to execute the experiment and gather empirical data. Should the hypothesis be validated, the subsequent phase would involve advancing to a phase 2 clinical trial in collaboration with experts in the field. Ultimately, the aim is to pioneer a novel treatment approach for Alzheimer's patients. Given that the most promising outcomes were achieved with the Pro-dermal model using injections at the Corpus Callosum and the Pre-clinical model with injections at the Hippocampus, it suggests that tailored treatment combinations may be necessary for different stages of Alzheimer's disease. This tailored approach could significantly enhance the efficacy and specificity of treatments, paving the way for more personalized care and improved patient outcomes.

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https://doi.org/10.1016/j.stemcr.2019.10.012