In vitro modeling of neurodegenerative diseases is now possible by using patient-derived induced pluripotent stem cells iPS. Through them, it is nowadays conceivable to obtain human neurons and glia, and study diseases cellular and molecular mechanisms, an attribute that was previously unavailable to any human condition. Amyotrophic lateral sclerosis ALS is one of the diseases that has gained a rapid advance with iPS technology. Here, we introduce a cellular platform to help maintain longevity of ALS iPS-motor neurons, a cellular feature relevant for most late-onset human diseases. Long term cultures of patient-derived iPS cells might prove to be critical for the development of personalized-drugs. Human iPS development.
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In vitro modeling of neurodegenerative diseases is now possible by using patient-derived induced pluripotent stem cells iPS. Through them, it is nowadays conceivable to obtain human neurons and glia, and study diseases cellular and molecular mechanisms, an attribute that was previously unavailable to any human condition. Amyotrophic lateral sclerosis ALS is one of the diseases that has gained a rapid advance with iPS technology.
Here, we introduce a cellular platform to help maintain longevity of ALS iPS-motor neurons, a cellular feature relevant for most late-onset human diseases. Long term cultures of patient-derived iPS cells might prove to be critical for the development of personalized-drugs. Human iPS development. A few years back , Yamanaka and colleagues Takahashi et al. They found that four transcription factors Oct4, Sox2, Klf4, and c-Myc were sufficient for transforming already mature human cells into a class of cells that had the ability to generate others, i.
This technological advance made possible a multiplicity of medical and scientific avenues, from generating specific human cells for regenerative medicine to cells that could be used for studying a diversity of human diseases Yu et al. Human iPS disease modelling. Most diseases are multifactorial, environmental or genetic or both factors must happen so the disease can be triggered.
Thus many diseases are not hereditary but sporadic in nature, which means that may or may not be genetic in origin, making its study tremendously complex. Despite this, there are sporadic diseases in which it has been possible to generate successful iPS model cells to study them Jung et al.
An example is amyotrophic lateral sclerosis ALS , a fatal neurodegenerative disorder that specifically affects spinal cord motor neurons which play a key role in motor function and respiration Almeida et al.
Therefore, it is imperative to have human models for studying mechanisms responsible for the disease onset and progression, and also for seeking development of specific therapeutic approaches. The modelling of neurodegenerative disorders with iPS cells might overcome several disadvantages of animal based- protocols Kehinde, ; Akhtar, , and give novel insights to the fields of human development and neurological diseases. ALS- human iPS motor neurons longevity. We and others have used iPS cells derived from patients to differentiate motor neurons Di Giorgio et al.
However, it is unclear how well newly derived motor neurons from iPS model late-onset diseases, where patients takes 50 or more years to develop ALS- symptoms. Here, we present a technical development to extend the longevity of iPS derived motor neurons. Our advance is relevant to the study of ALS, but it can also be implemented to other late- onset diseases to address the role of age-associated factors. Mouse genetic models and derivation of glia. Mice were handled in accordance to the guidelines established by the bioethics committee of University of Talca, following approved protocols for the good care and use of laboratory animals.
Under a dissecting microscope, tissue was isolated, cut into small pieces and transferred to 50 ml centrifuge tube containing 12 ml of HBSS. Once confluent generally in d , cells were replated onto 12 well dishes coated with PDL Sigma.
Culture of iPS lines and Differentiation to motor neurons. To generate motor neurons, undifferentiated iPS cells were incubated with 10 mM Rho-associated kinase inhibitor Y Calbiochem for 2hr and then passaged using trypsin, triturated, and placed into ultra-low adherent culture dishes Corning for the seeding of embryoid bodies EBs. Finally, at day 28, EBs were dissociated with 0. To prepare cells for live imaging, they were allowed to settle on glass coverslips that were flipped over primary glial monolayers to sustain neuronal development and maturity.
Media was changed every other day and all iPS derived motor neurons iMNs were kept for a period of weeks. Immunocytochemical experiments. Then, they were incubated overnight with the following antibodies for survival studies: rabbit polyclonal anti- Neurofilament Chemicon , and mouse anti-HB9 Developmental Studies Hybridoma Bank University of Iowa.
Samples were then mounted in Vectashield Vector Labs , and confocal or epifluorescence microscopy was performed using LSM Zeiss , equipped with 20, 40 and 60X oil immersion objective, 1. Image acquisition was performed using Zen software Zeiss. Offline analysis of relative intensities for all the samples were done using Metamorph 4.
Only cells having morphological features of neurons were considered for analysis. Invitrogen Catalog Numbers A and A Data analysis. To reduce variability, experiments were performed in sister cultures. Experimental design to culture human ALS- motor neurons for long periods of time. Our goal is to understand the mechanisms that trigger ALS disease.
For this, we used human motor neurons derived from iPS cells. Typically, these cells do not survive for more than 14 days in vitro when cultured under traditional single cell culture. As we indicated earlier, ALS is a late onset disease, thus much of the phenotype is likely to be absent in 14 days of in vitro development. To overcome this limitation of the model, we developed a cellular methodology to lengthen the lifespan of ALS- motor neurons in vitro Fig.
These cellular formations were then treated with retinoic acid RA and purmorphamine PUR to induce caudalization and ventralization of pluripotent stem cells, here we are directing the fate of these stem cells into a motor neuron. Traditionally, motor neurons differentiated from neurospheres are plated on polystyrene petri dishes and survive at most for about fourteen days in vitro. To elongate the lifespan of the cells we first plated them on clean glass treated with a combination of laminine and poly-d-lysine, after these initial treatment we use drops of wax to form a tripod in the glass see methods.
This step is very useful if the motor neuron population is desired to be studied independently Phatnani et al. In parallel, we prepared a culture of mouse or human glial cells when needed, which may or may not be ALS positive.
Glial cells were then plated on glass and kept in vitro for 2 weeks until the cells reach confluency. After this period the motor neurons derived from IPSs are platted on the glass as previously described and flip over the confluent monolayer of Glia.
The diagram shows a co-culture system, specially designed to study the interactions between neurons and Glia. We adapted this model from a commonly used technique to culture hippocampal neurons for long term. First, iPS cells were grown in colonies A , later they were seeded in suspension to form embryoid bodies B and go through the differentiation protocol C-E.
After 4 weeks, human motor neurons were plated over glass coverslips previously treated E and flipped over Glia monolayers. In parallel, Glia monolayers were cultured from neonatal mouse P0-P3 F , using brain cortex G to extract glial precursors H that reached confluency at 2 weeks in vitro i. The resulting sandwich coculture system is shown in J.
From iPS to long-term cultures of motor neurons. To demonstrate pluripotency of iPSs a panel for trophic factor markers was used. As we expected, the iPS cells were positive for Nanog Fig.
The pluripotency of the cells was confirmed by the teratoma formation assay, and the karyotypes remained stable in all passages used Bilican et al. The iPSs derived motor neurons iMNs were generated by using a chemical protocol methods. We then plated iPS cell-derived motor neurons together with primary cortical glial cultures derived from wild type neonatal mice and cultured the cells for 3, 7, 14, 28 days in vitro or longer two months.
Specifically, the iPSs- derived motor neurons were plated on glass coverslips coated with poly-d-lysine and laminin; and inverted over wells containing glial cells monolayers.
Motor neurons survived for long periods of time, from 5 days to more than 40 days, and some cases for 60 days in vitro. In general, cells at 21 days of age were showing morphological hallmarks of a mature neuron, including enlarge soma i. A representative brightfield image of an iPS colony is shown in F. A-F show representative images of IPS derived motor neurons. Images in A, B,C,D and E show neurons cultivated for 5, 7, 14, 21 and 28 days respectively; F shows a diagram of the neurons as represented in Figure 1.
The Sandwich approach to elongate the in vitro life of iPSmotor neurons. Here, the long term grown cells sandwich of glial cells , were stained with two well known molecular markers, neurofilament a classic neuronal marker and HB9 motor neuron specific marker. Irrespective of the in vitro age analyzed, a dense network of cellular processes was observed in all samples studied, a condition that demonstrates the healthy state of neurons grown on the sandwich format.
To establish that neurons were indeed motor neurons, we used an antibody against HB9. Overall, we show that ALS- patient derived motor neurons can be maintained in vitro for more than 40 days. Images were acquired with a 40x NA1. It is evident that the phenotypes and classic hallmark pathologies are not fully represented in-vitro. The reasons for this are not really clear, but may be associated to the fact that neurons generated from iPS may be too young in the dish, compared to those present in patients with late onset diseases.
We and others have showed evidence that iPSC-derived motor neurons can model early changes of ALS- disease, and thus prove the utility of the method to study disease mechanisms.
If however, we want to characterize ALSclassic hallmarks in these cells, we find that many of them are not present in the cultures i. It might be that cultures need to be maintained for longer to fully represent a late onset disease, and thus by extending the lifespan of the cultures we might be able to reveal the pathological hallmark of patients in the in-vitro model.
One approach to extend the life span of cells is presented here. We developed a cellular based approach to produce co-cultures of glial and human iPSmotor neurons; the iPS- motor neurons were plated on glass coverslips to face a monolayer of astrocytes.
In general, motor neurons were more mature, they have a large cell body connected with an intricate network of processes, with distinctive physiological properties.
The two cell types are maintained physically apart, although they share the same medium. Thus, factors secreted by one cell type likely impact on the health, survival, gene expression of the other, a process that we and others are very interested to analyze because molecules from cells in particular astrocytes might negatively interfere with the other cell. These non-cell autonomous interactions play a crucial role in several conditions of ALS disease development.
Future avenues. The co culture method offers the possibility for understanding the contribution of cell-autonomous and non-cell autonomous mechanisms to the ALS. We have used this setup for RNA sequencing individual cell types model to study molecular pathways specific to each cell type. Also, the long term cultures may be especially useful for drug screening, for studying for high-throughput drug and effector screens. Clearly, the use of long term cultures is a major improvement in our understanding of late onset neurodegeneration.
Despite all, it is worth keeping in mind that the model might not fully represent the situation found in patients. Our approach could also be used for growing cells for cellular therapies to treat diseases or injuries.
Cellular replacement for neurodegenerative diseases involves the derivation of specific neuronal subtypes lost in disease and subsequent grafting into affected areas of the nervous system. The newly transplanted neurons may then integrate, synapse and recapitulate a neural network similar to the one lost in disease. Alternatively, stem cells may provide environmental enrichment to support host neurons by producing neurotrophic factors, scavenging toxic factors or creating auxiliary neural networks around affected areas.
Many strategies for environmental enrichment utilize stem cells to provide de novo synthesis and delivery of neuroprotective growth factors at the site of disease. The appropriate objective of cellular therapy for each neurodegenerative disease must be based on the specific neuronal pathology of each disorder.
2010, Número 591
DOI Its etiology is still not clear. Worldwide, prevalence ranges from 2 to 11 cases per , people. Age of presentation varies from 58 to 63 years for sporadic cases, and from 47 to 52 years for the familial ones.
Asociación Colombiana de Esclerosis Lateral Amiotrófica
Please donate. Skip to primary navigation Skip to main content Skip to primary sidebar Skip to footer. Email Facebook Twitter. Search this website. To achieve its goals, the organization will perform following specific activities: To organize Medical and practical seminars for patients, family members of patients and caregivers To coordinate activities with medical organizations and ALS research institutes To promote the appropriate and dignified medical care for ALS patients To raise donations for the progression of the Association To establish and maintain relationships with International ALS Associations and with national and foreign organizations with similar purposes To promote the creation of ALS chapters in other cities To advise patients and family members regarding the health rights and related Constitutional rights To find psychological support for patients and family members To make the society sensitive about the devastating effects of the disease.
Amyotrophic lateral sclerosis
Amyotrophic lateral sclerosis : update. Iatreia [online]. ISSN Its etiology is still not clear.