A new protocol allows researchers to generate spinal motor neurons derived from Charcot-Marie-Tooth disease (CMT) patients, creating useful models for better understanding the disease and investigating responses to potential therapies.
The protocol was described in “Optimized Protocol to Generate Spinal Motor Neuron Cells from Induced Pluripotent Stem Cells from Charcot Marie Tooth Patients,” published in Brain Sciences.
Recent advances in genetic technologies have greatly expanded understanding of the mutations that underlie CMT. However, understanding the consequences of these mutations at the level of cells and organisms is challenging. While animal studies can be useful in this regard, they are not always feasible, for both logistical and ethical reasons.
An alternative is studying cells in dishes, called in vitro studies. By definition, such studies require access to the right types of cells. In the case of CMT, this typically involves nerve cells such as spinal motor neurons, important for sending signals from the brain to muscles.
Because spinal motor neurons cannot come directly from patients, human induced pluripotent stem cells (hiPSCs), which can be generated from blood or skin cells, are seen as a helpful alternative. As their name suggests, these cells are pluripotent — meaning that, like other stem cells, they are able to differentiate into other cell types.
As such, researchers can collect blood or skin cells from a person with CMT and use these to generate hiPSCs, which can then be differentiated into spinal motor neurons. Since these motor neurons are derived from the hiPSCs of a CMT patient, they will have the same genetic abnormalities, making them an apt model for study.
This process involves giving cells the correct biochemical cues. While multiple protocols have been developed for generating motor neurons from hiPSCs, they are often ineffective or inefficient when using cells from people with genetic diseases like CMT.
Researchers in France devised a protocol for the hiPSC-mediated generation of spinal motor neurons using cells from two people with CMT type 2 (CMT2) and from five people without CMT.
The hiPSCs were first developed using a previously established protocol. “It was more difficult to obtain the hiPSCs from the CMT2 patients than the controls,” the researchers wrote. “Nevertheless, it was possible to obtain hiPSCs from both.”
Their protocol for generating spinal motor neurons from the hiPSCs was devised after trying several previous protocols, with limited success. The researchers gave consideration to how motor neurons usually form during development in designing their protocol, and several different protocols were tested.
Broadly, this involved trial-and-error concerning the type, timing, and amounts of various growth factors and other signaling molecules that regulate cell growth and differentiation. A specific protocol ultimately was able to create motor neurons within 20 days, with a success rate of 80%.
In early examinations of the generated motor neurons, the researchers noted changes in the CMT motor neurons, such as stronger inward currents and weaker outward currents, which have been previously reported in CMT neurons. These data support the use of motor neurons generated through this protocol as a cell model of CMT.
“This protocol should aid researchers to easily and rapidly differentiate hiPSCs into [motor neurons] … using a limited number of growth factors, with a high success rate,” the researchers wrote.
“These models will allow investigation of the molecular pathways involved in the disease and, hopefully, help in the development of new therapeutic strategies, particularly as a tool for drug screening,” they concluded.
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