D Cell Culture Plating
For 3D seeding, cells were washed with PBS. Warm Axol Unlock XF Media was added to the cells for 3 min at 37 °C for the cells to detach. Cells were recovered in ANM media and spun at 200 g for 5 min. The pellet was resuspended in fresh warm ANM media. Cells were plated on ice, in an 8-well chamber slide at a density of 2×106/ml in 3D plating media composed of ANM media with Retinoic Acid at 500 ng/ml and supplemented with Matrigel at a ratio of 1:15 v/v, and then left to form a gel at 37 °C. An extra 200 µl of ANM media was added 2 h after the cultures were plated.
Quantseq 3 Mrna Sequencing Data Analysis
The QuantSeq 3â² mRNA-Seq reads were aligned using Bowtie2 . Bowtie2 indices were either generated from the genome assembly sequence or the representative transcript sequences for aligning with the genome and transcriptome. The aligned file was used to assemble the transcripts, estimate their abundance, and detect the differential expression of genes. Differentially expressed genes were determined based on unique counts and multiple alignments using Bedtools . The RT data were processed based on the quantile-quantile normalization method using EdgeR within R software using Bioconductor . Gene classification was based on searches conducted in the DAVID and Medline databases .
Sgrn Is The Main Effector Of Primed Mscs
To confirm whether SGRN was the main inducer of primed MSCs, various concentrations of SGRN protein were used to treat naïve MSCs . After the treatment of the naïve MSCs with SGRN for 24h, the TGF-β mRNA expression in naïve MSCs was measured via quantitative real-time PCR analysis. Significant increases in TGF-β mRNA expression were observed, except at 10ng/mL ). The peak was observed at 2mg/mL SGRN treatment. Next, the therapeutic potential of SGRN-treated MSCs was briefly assessed ). H4SW cells were cocultured with naïve MSCs or SRGN MSCs for 24h, and then, the CCK assay was conducted to confirm the antiapoptotic effect of naïve and SRGN MSCs on H4SW cells. Cell death was significantly inhibited when H4SW cells were cocultured with naïve MSCs and SGRN MSCs. This suggests that SGRN secreted by H4SW cells or the AD microenvironment is an inducer of primed MSCs.
Effects of SRGN-primed MSCs. The mRNA was extracted from SRGN-primed MSCs and analyzed by qPCR using a specific primer for human TGF-β. âp< 0.05, n = 3. Viability of H4SW cells under serum-starved conditions analyzed by CCK-8 assays. Three samples per experimental group were tested in each assay. The data are presented as the mean ± S.E.M. âp< 0.05.
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Primed Mscs Show In Vitro Therapeutic Effects Against Alzheimer’s Disease
Next, we performed Western blot analysis to confirm the therapeutic efficacy of primed MSCs on AD pathology, especially Aβ and ubiquitin conjugates. Aβ is the most well-known pathological hallmark of AD. Ubiquitin conjugates are negatively correlated with 26S proteasome activity, which means that impaired 26S proteasome activity results in the accumulation of Aβ, hyperphosphorylated tau, and ubiquitin conjugates in the AD brain. Therefore, along with Aβ, the level of ubiquitin conjugates was measured in this study as another hallmark of AD.
Therapeutic effect on Alzheimer’s disease in vitro. Secreted amyloid-beta levels in the conditioned media were measured by ELISA. âp< 0.05. The degradation of ubiquitin conjugates in H4SW cells with cocultured MSCs was measured by Western blots. The percentage of Ub conjugates was calculated as a percentage of that in the H4SW cells. Changes in mRNA expression were evaluated. The data are presented as the mean ± S.E.M. Three samples per experimental group were tested in each assay. âp< 0.05.
In addition, the differences in gene expression in H4 and H4SW cells cocultured with naïve MSCs or primed MSCs were analyzed. The analysis revealed that the dysregulated genes in the H4SW AD in vitro model were altered toward normal conditions after coculture with naïve MSCs and primed MSCs. Between the two MSCs, primed MSCs showed better alteration .
Current Status And Challenges Of Stem Cell Treatment For Alzheimers Disease
Article type: Review Article
Authors: Pacheco-Herrero, Mara * | Soto-Rojas, Luis O.b | Reyes-Sabater, Heidya | Garcés-Ramirez, Lindac | de la Cruz López, Fidelc | Villanueva-Fierro, Ignaciod | Luna-Muñoz, Josée f *
Affiliations: Neuroscience Research Laboratory, Faculty of Health Sciences, Pontificia Universidad Católica Madre y Maestra, Dominican Republic | Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, State of Mexico, Mexico | Escuela Nacional de Ciencias Biológicas, Depto de Fisiología, Instituto Politécnico Nacional, Mexico City, Mexico | Instituto Politécnico Nacional, CIIDIR, Unidad Durango, Durango, México | National Dementia BioBank, Ciencias Biológicas, Facultad de Estudios Superiores Cuautitlán, UNAM, State of Mexico, Mexico | Banco Nacional de Cerebros-UNPHU, Universidad Nacional Pedro Henríquez Ureña, Dominican Republic
Correspondence: Correspondence to: Mar Pacheco-Herrero, Neuroscience Research Laboratory, Faculty of Health Sciences, Pontificia Universidad Católica Madre y Maestra, Dominican Republic. E-mail: José Luna-Muñoz, National Dementia BioBank, Ciencias Biológicas, Facultad de Estudios Superiores Cuautitlán, UNAM, State of Mexico, Mexico. E-mail: .
Keywords: Alzheimers disease, amyloid-, neural stem cells, neurodegeneration, stem cells, tau protein, therapy
DOI: 10.3233/JAD-200863
Journal: Journal of Alzheimer’s Disease, vol. 84, no. 3, pp. 917-935, 2021
Abstract
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Mesenchymal Stem Cell Therapy For Alzheimers Disease
Abstract
Alzheimers disease is a neurodegenerative disease responsible for 60-70% of the 50 million cases of dementia worldwide. It is characterized by neuronal cell death, shrinkage of brain tissue, and progressive cognitive, motor, and behavioral impairment, which often leads to death. Although current treatment has helped improve the patients quality of life, it has not been able to alter the underlying disease pathology of AD. Studies have shown that mesenchymal stem cells a group of multipotent stem cellshave the ability to stimulate neuroregeneration and inhibit disease progression. More recently, extracellular vesicles from cytokine-preconditioned MSCs have also shown to induce immunomodulatory and neuroprotective effects in AD models. This review will aim to compile pertinent preclinical AD research on transgenic mice as well as clinical trials on MSC-based therapy from diverse sources.
1. Introduction
2. Pathogenesis of AD and Current Treatment
3. MSC as an Alternative Treatment
4. Preclinical Studies
5. Clinical Studies
Many clinical trials have been conducted to evaluate the role of MSCS derived from various sources in patients with AD. It is important to note that only a few have been officially completed and have published results. A summary of the clinical trials registered on ClinicalTrials.gov evaluating MSCs for AD treatment can be found in Table 2.
5.1. Completed
5.2. Ongoing
5.3. Challenges
6. Conclusions
Data Availability
Conflicts of Interest
Alzheimers Disease: How Could Stem Cells Help
Alzheimers disease is the most common cause of dementia. It is a complex disease that affects nerve cells in many parts of the brain, making effective treatment very challenging. Can stem cell research help us tackle this challenge in the future?
Alzheimers disease is the leading cause of dementia. People affected by AD commonly experience memory loss, confusion and mood swings.
The cause of AD is still unknown, but several theories focus on two proteins, called amyloid beta and tau, which are found in deteriorating areas of an AD brain.
Clumps of amyloid beta proteins form plaques that may prevent neurons from sending signals properly.
Tau protein is important for normal cell function, but researchers think that when tau gets gnarled up into tau tangles it prevents neurons from getting nutrition.
There is currently no cure for AD.
No stem cell treatments are currently approved for AD. Positive effects have been seen with neural stem cell transplants given to mice with a disease similar to AD, but researchers are still studying what these stem cells are doing and how they might help repair the brain.
Researchers are using induced pluripotent stem cells to grow neurons that have the same genetic background as people affected by AD so they can study the disease. These neurons represent a tool to look for new drugs that can reduce amyloid and tau, and also find disease signposts that can help diagnose patients with AD earlier.
Plaques and tangles:
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The Evaluation Of Therapeutic Effect
Stem cells such as NSCs, BMMSCs, hUCBMSCs, ESCs, and iPSCs have been investigated in different ADlike animal models. Furthermore, hUCBMSCs, hPDMSCs, hBMMSCs, and hAD SVF are being tested in different clinical trials. The evaluation of therapeutic efficacy involves behavioral performance tests in animal models, and biochemical and pathohistological indicators. Examples of behavioral performance tests include Morris water maze, Barnes maze, Ymaze, Tmaze, zeromaze, 8arm maze, plusmaze discriminative avoidance task, shuttle box test, step down test, open field test, and dark avoidance.59, 81 In clinical trials, the diseaserelated severity of all subjects is evaluated based on symptoms, cognitive function, memory, and quality of life. Biochemical and pathohistological changes of the APP/PS/tau triple transgenic model are informative in assessing coevolving amyloid and tau pathologies, which are related to the pathomechanism of Alzheimer’s disease.82 The pathophysiological changes of A and tau in the human brain occur before the onset of AD symptoms. There are high levels of A42, ttau, and ptau in peripheral neurogenic exosomes and cerebrospinal fluid, which is powerful evidence for the diagnosis of AD.83, 84.
Traditional Treatment For Alzheimer’s In 2022
- Multiple specialties: geriatrics, general medicine, neurologist, psychologist, physical therapist.
- Pharmaceutical drugs to increase cognitive enhancement.
- Medication to lower blood pressure and to prevent intracranial clots.
- Antipsychotic drugs to balance mood and reduce uncooperativeness.
- Physical exercise to improve cardiovascular strength and promote healthy brain synapses.
- Hospice care is sought in advanced stages as the condition worsens.
- Cost ranges from $10,400 to $34,517 USD per patient annually in MCOs. Costs increase as the disease worsens year after year.
Because there is no known cure for Alzheimers, and it worsens over time, treatment typically consists of cognitive enhancing medication and symptom management. Due to the fact that it is very difficult for an adult patient to generate new healthy brain cells in large quantities fast enough to counteract progression of the disease, the objective of this medical approach is to provide temporary relief to patients.
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Mesenchymal And Hematopoietic Stem Cells
Among the different ASC lines, mesenchymal and hematopoietic stem cells can be easily obtained and thus have been the ones most widely studied as possible therapeutics for AD. MSCs are hierarchical postnatal stem cells, capable of self-renewing and retaining diverse differentiation potency . MSCs may be isolated from multiple sources and can differentiate into multiple types of tissue, have high expansion capacity and low immunogenicity and carcinogenic potential . Concerning their effects in AD pathology, MSCs have been reported to enhance neurogenesis through the release of neurotrophic factors, enhance A clearance, modify innate and adaptive immune cell responses through upregulating neuroprotective cytokines such as IL-10 while decreasing the amount of pro-inflammatory cytokines TNF- and IL-1 . Reduction in amyloid-beta deposition and increased microglial phagocytic activity has also been observed .
How Can Stem Cells Help Patients With Alzheimers Disease
Apr 15, 2020 | Alzheimers Disease, Stem Cell Therapy
As the most common cause of dementia, Alzheimers disease affects hundreds of thousands of people in the U.S. Its a progressive brain disease which impairs cognition, including memory, behaviors, and thinking. Over time, symptoms worsen and begin to interfere with a persons ability to perform daily activities.
In people with Alzheimers, plaques, or protein deposits, form between nerve cells at a quicker rate, as do tangles, or twisted fibers which accumulate within dead nerve cells. As a result, these damaged nerve cells are unable to transmit necessary electrical signals.
Currently, there is no cure for Alzheimers, though some treatments can help to control certain symptoms. In the quest to find a more effective treatment to slow the progression of the disease and potentially even improve symptoms in patients, researchers are turning to regenerative medicine therapies, including stem cell treatment.
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Central Role Of Neuroinflammation In Alzheimers Disease
Neuroinflammation plays a central role in pathogenesis of AD . According to the classical view, neuroinflammation is characterized by activated microglia and reactive gliosis surrounding the amyloid plaques in AD . In this theory, neuroinflammation is considered a passive reaction toward amyloid plaque and Tau protein. Recent studies suggest neuroinflammation actually precedes AD classical hallmarks, which means neuroinflammation is in itself a contributor to AD pathogenesis and represents as the third pathological hallmarks of AD . Thus, the neuroinflammation theory has evolved. A recent review by Cuello best summarizes this new paradigm shift thinking for neuroinflammation in AD. AD in pathological view needs to be considered as a continuum of disease in which neuroinflammation can be divided into two phases: the early phase and the late phase . The early neuroinflammation phase is the long prodromal phase in AD . In this early neuroinflammation phase, a disease-aggravating CNS inflammation predominates and microglia exhibit a pro-inflammatory profile In late neuroinflammation phase which represents the phase after AD clinical manifestation, the neuroinflammation wanes down to great extent.
The central player of neuroinflammation is microglia, however, the importance of astrocytes and other immune cells have gained increased attention in recent years.
Evaluation Of Therapeutic Efficacy Of Primed Mscs In 5xfad Mice
To evaluate the efficacy of primed MSCs in AD, we performed an in vivo experiment using a 5xFAD AD transgenic mouse. The experimental animals were divided into four groups: wild-type control , transgenic control , naïve-MSC, and primed MSC. We injected 1 à 105 WJ-MSCs into the right lateral ventricle. One week after injection, the mice were euthanized, and brain tissues were harvested. First, the antiapoptotic effect of primed MSCs was assessed by cleaved caspase-3 Western blot analysis ). When compared to the WT mice, the 5xFAD mice showed increases in cleaved caspase-3, indicating neuronal death in the brain, whereas both the naïve MSCs and primed MSCs significantly reduced cleaved caspase-3 levels in the brain. Next, Aβ accumulation in the brain was measured by Western blots and thioflavin-S staining ). Compared to the WT control, the deposition of Aβ in the brain was observed in 5xFAD mice. The groups injected with naïve MSCs and primed MSCs showed decreases in Aβ accumulation. Primed MSCs, in particular, attenuated Aβ accumulation more effectively than naïve MSCs, which was confirmed by thioflavin-S staining. Thioflavin-S staining ) revealed extensive Aβ deposits in the cortex and hippocampal regions of the 5xFAD transgenic mouse control group . Strikingly, the amount of Aβ in the cortex and hippocampus was reduced in the groups injected with naïve and primed MSCs, and the primed MSC group showed better therapeutic efficacy.
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Advances In Stem Cell Therapy In Alzheimers Disease: A Comprehensive Clinical Trial Review
Nikolaos Karvelas1#^, Samuel Bennett2#^, Georgios Politis1, Nikolaos-Iasonas Kouris1^, Christo Kole1^
1 Faculty of Medicine, National and Kapodistrian University of Athens, Athina , Emory University School of Medicine , , USA
Contributions: Conception and design: C Kole Administrative support: NI Kouris Provision of study materials or patients: N Karvelas, S Bennett, C Kole Collection and assembly of data: N Karvelas, S Bennett, G Politis, NI Kouris Data analysis and interpretation: N Karvelas, S Bennett, C Kole Manuscript writing: All authors Final approval of manuscript: All authors.
#These authors contributed equally in this work.
^ORCID: Nikolaos Karvelas, 0000-0003-1452-1192 Samuel Bennett, 0000-0002-4514-4388 Nikolaos-Iasonas Kouris, 0000-0002-1659-075X Christo Kole, 0000-0003-4695-3723.
Correspondence to:
Keywords: Alzheimers disease stem cell therapy embryonic stem cells mesenchymal stem cells induced pluripotent stem cells
Received: 11 November 2021 Accepted: 27 January 2022 Published: 21 February 2022.
doi: 10.21037/sci-2021-063
Limitations Of Current Mesenchymal Stem Cells Trials For Alzheimers Disease
Despite the safety being demonstrated in MSCs clinical trials, efficacy has not been proven. We must keep in mind that by the time AD is clinically diagnosed, the neuronal loss and pathological proteins have already accumulated in many brain regions and therefore it is difficult to reverse the disease process. Furthermore, in many clinical trial protocols, patients may receive only several times of stem cells infusion, whereas they indeed might need multiple stem cells infusions over extended period of time. In some trials, autologous MSCs were used. AD patients are usually advanced in age and autologous MSCs may suffer from senescence which compromises their regeneration capability. Most AD clinical trials used intravenous route. The vast majority of intravenously administered stem cells will get detained in the lung and the spleen and only very limited number of stem cells can get into the brain. The hostile micro-environment also hindered the survival of infused stem cells. As a result, the paracrine effects of exogenous stem cells cant compensate for the vast loss of majority neuronal cells in the patient. Anyway, there are many limitations for current MSCs clinical trials which await immediate renovation in the field.
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Endogenous Neural Progenitor/stem Cells
Since neural progenitor/stem cells have been proven to exist in the adult CNS and to be involved in the neurogenesis process, the activation of endogenous neural progenitor/stem cells populations that can migrate to the injured regions, proliferate and functionally integrate into the existing circuit represents a significant strategy to promote neural regeneration in the diseased brain. This activation within the brain is to protect the remaining tissues and prevent secondary neuron loss through the production of neurotrophic and neuroprotective factors, such as brain derived neuronal factor and vascular endothelial growth factor. A recent research has demonstrated that the self-repair in the adult brain can be augmented by the infusion of growth factors to activate endogenous neural precursor cells that contribute to new tissue formation and functional recovery after stroke.