Building blocks are defined as the basic units from which something is built up. Since a finished product is only as good as the foundation upon which it is laid, the building blocks must be of the highest quality in order for the final goal to be accomplished. This is also true in molecular biology. Molecular biology building blocks are the everyday reagents that are used as part of an experiment. Without high-quality reagents, the final result of the experiment is compromised.
Take, for example, the work of Samantha Butler’s laboratory at the University of California Los Angeles: their primary focus is on repairing spinal cord injuries through neuroregenerative treatments using stem cells. Dr. Sandeep Gupta, a researcher in Butler’s laboratory, works to understand the basic mechanisms behind how the spinal cord is formed during embryo development, and how that can apply to therapies for spinal cord injury.
Dr. Sandeep Gupta
Sandeep received his PhD in 2015 from the Indian Institute of Technology, Kanpur (India). His research in the Butler lab at UCLA is focused on understanding how bone morphogenic protein (BMP) signaling performs diverse functions such as patterning and regulating neuronal growth in the dorsal spinal cord.
Any sort of damage to the spinal cord that can impair its function is considered a spinal cord injury. This impairment can be temporary or permanent, although most often it is a permanent situation. Spinal cord injuries are estimated to affect over a million people in the United States alone. More than 2,000 permanent spinal cord injuries occur each year, the majority of which are seen in military personnel. These conditions have a significant impact on our society, for both the medical personnel treating the injuries (at a cost of $40 billion annually) as well as for the caretakers of those individuals. Not only do patients lose movement, but they also lose sensation, which causes a disconnect from their environment and a reduction in their quality of life.
We currently don’t have any means to reverse spinal injuries. Available methods mainly utilize the neuroprotective properties of certain chemicals and proteins that facilitate blood flow to the injured area and limit further damage. However, a proposed option is neuroregenerative, where the focus is on the regeneration of the lost neurons. This goal is typically accomplished through exercise or through the application of specialized cells: neural stem cells that can actually build the lost neurons inside the body. My research focus is to investigate how this can be achieved.
Significant progress has been made generating the in vitro–derived motor neurons required to restore coordinated movement. While these motor neurons are important, they do not address the problem of sensation. Your body needs constant feedback to guide the motor system to function. For example, if you cannot sense pain, you cannot guide your motor neurons to avoid the source of the pain. Movement cannot function without sensory information, which is why we are focused on understanding the molecular mechanism of sensory interneuron differentiation to define ways to convert induced pluripotent cells (iPSCs) into sensory neurons.
We have a multitiered approach to our research. To identify what new genes are involved in the differentiation of stem cells into spinal cord neurons, our initial discovery strategy uses RNA-Seq. Once those genes have been identified, we then use RT-qPCR to confirm that they are not due to experimental error. We consider RT-qPCR to be one of the most important tools that we use every day. This technique helps to confirm that differentiation is proceeding correctly by detecting markers at certain timepoints during the process. Assaying these markers through RT-qPCR is an easy, high-throughput, and robust method. It is also the first line of investigation used to check if differentiation has improved with the inhibition or activation of certain signaling pathways. It is very important to use RT-qPCR in my research to help successful monitoring of the differentiation process.
We run 6–12 samples at a time every day. Therefore, speed is of high importance to reduce processing time. We also need a high-fidelity system that will consistently produce cDNA copies of any long RNA templates. This was a problem, until we began using Invitrogen SuperScript IV Reverse Transcriptase. The SuperScript IV reagent is very fast and efficient. We’re able to produce a much higher cDNA yield in 20 minutes, vs. 50 minutes per reaction previously. With SuperScript IV reagent, we’re consistently producing results using long RNA templates, which was difficult before.
iPSCs are remarkable tools, since you generate the cells directly from the patient. In doing so, there is no risk for rejection of the cells when transplanted and the patient can receive the maximum benefit of the treatment. Before we can use this approach, however, we still have a long way to go with the research. We don’t know how these neurons are born in the spinal cord. The neurons in the spinal cord are connected in a very precise way. We still do not understand how they connect and how they find their partner for a correct function. To restore spinal cord injuries, we have to figure out how to make these spinal cord neurons in a dish and test if they can make a desired connection when putting them back in the injured spine. They can also be an excellent platform to screen potential drugs that might be effective for growing the axons for a particular neuron.
As you can see from Dr. Gupta’s research, knowledge of how neurons are first developed is crucial to creating these cells in a clinically relevant setting that can then be used for neuroregenerative spinal cord stem cell treatment. It is the foundation for this therapy; and to ensure that the proper building blocks are being laid, Dr. Gupta trusts the initial investigation of his research to high-quality molecular biology reagents for his experiments.
Learn more about Dr. Gupta and the work that Dr. Butler’s laboratory is accomplishing
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