Researchers at MIT have achieved a significant breakthrough in regenerative medicine, creating a streamlined method to transform skin cells directly into neurons. This innovative approach bypasses the need for an intermediate stem cell stage, offering a more efficient pathway for generating neurons. The findings are detailed in two papers published in the journal *Cell Systems*.

The research team, led by Katie Galloway, the W. M. Keck career Advancement Professor in Biomedical Engineering and Chemical Engineering, developed the conversion method using mouse cells. The process is remarkably efficient, capable of producing more than 10 neurons from a single skin cell. Replicating this success in human cells could revolutionize the large-scale production of motor neurons for therapeutic applications.

Traditionally, converting one cell type to another involves inducing the skin cell into a “pluripotent” stem cell, which is then differentiated into the desired cell type, such as a neuron. This multi-step process can be time-consuming and less efficient. The MIT researchers’ direct conversion method offers a more direct and potentially faster route.

According to the research, a key aspect of this breakthrough is the potential for cell replacement therapies.

We where able to get to yields where we could ask questions about whether these cells can be viable candidates for the cell replacement therapies, which we hope they could be. That’s where these types of reprogramming technologies can take us.

Katie Galloway, the W. M. Keck Career Development Professor in Biomedical engineering and Chemical engineering

As a crucial step toward therapeutic request, the researchers demonstrated the successful generation of motor neurons and their engraftment into the brains of mice.These transplanted neurons integrated with the host tissue, suggesting their potential to restore lost function.

The two *Cell Systems* papers detailing this new method list Galloway as the senior author, with MIT graduate student Nathan Wang as the lead author of both.

the Evolution of Cell Conversion: From iPSCs to Direct Conversion

The journey to this breakthrough began nearly 20 years ago when scientists in Japan discovered that delivering four transcription factors to skin cells could induce them to become induced pluripotent stem cells (iPSCs). Similar to embryonic stem cells, iPSCs possess the remarkable ability to differentiate into various cell types. While this technique proved effective,it typically requires several weeks,and a significant portion of the cells fail to fully transition into mature cell types.

Galloway explains the challenges associated with traditional reprogramming methods:

Oftentimes, one of the challenges in reprogramming is that cells can get stuck in intermediate states. So, we’re using direct conversion, where instead of going through an iPSC intermediate, we’re going directly from a somatic cell to a motor neuron.

Katie Galloway,the W. M. Keck Career Advancement Professor in Biomedical Engineering and Chemical engineering

While Galloway’s research group and others have previously explored direct conversion,the yields were historically low,often less than 1 percent. Galloway’s earlier work involved a combination of six transcription factors and two proteins that stimulate cell proliferation, delivered using separate viral vectors. This complex delivery system made it challenging to ensure consistent expression levels of each gene in every cell.

The first of the new *Cell Systems* papers details the streamlined process developed by Galloway and her students. This improved method converts skin cells into motor neurons using only three transcription factors, along with the two genes that promote cell proliferation.

The researchers began with the original six transcription factors and systematically eliminated them,one at a time,until they identified a combination of three – NGN2,ISL1,and LHX3 – that could successfully complete the conversion to neurons in mouse cells.

Reducing the number of genes to three allowed the researchers to use a single modified virus to deliver all three transcription factors, ensuring consistent expression levels in each cell.

A separate virus was used to deliver genes encoding p53DD and a mutated version of HRAS. These genes stimulate the skin cells to divide rapidly before converting to neurons, resulting in a considerably higher neuron yield, approximately 1,100 percent.

Galloway elaborated on the importance of cell proliferation in the conversion process:

If you were to express the transcription factors at really high levels in nonproliferative cells, the reprogramming rates would be really low, but hyperproliferative cells are more receptive. It’s like they’ve been potentiated for conversion, and then they become much more receptive to the levels of the transcription factors.

Katie Galloway, the W. M. Keck Career Advancement Professor in Biomedical Engineering and Chemical engineering

The researchers also developed a slightly different combination of transcription factors that enabled them to perform the same direct conversion using human cells, although with a lower efficiency rate, estimated between 10 and 30 percent.This process takes approximately five weeks, which is slightly faster than the traditional method of converting cells to iPSCs and then into neurons.

Optimizing Delivery and Implanting Converted Cells

Having identified the optimal gene combination, the researchers focused on optimizing the delivery methods, as described in the second *Cell Systems* paper.

They tested three different delivery viruses and found that a retrovirus achieved the most efficient conversion rate. Reducing the density of cells grown in the dish also contributed to improving the overall yield of motor neurons. This optimized process, which takes about two weeks in mouse cells, achieved a yield exceeding 1,000 percent.

In collaboration with colleagues at Boston University, the researchers assessed the success of engrafting these motor neurons into mice.The cells were delivered to the striatum, a region of the brain involved in motor control and other functions.

After two weeks, the researchers observed that many of the neurons had survived and appeared to be forming connections with other brain cells. When grown in a dish, these cells exhibited measurable electrical activity and calcium signaling, indicating their ability to communicate with other neurons. the researchers are now exploring the possibility of implanting these neurons into the spinal cord.

The MIT team aims to further improve the efficiency of this process for human cell conversion. This advancement could pave the way for generating large quantities of neurons for treating spinal cord injuries or diseases affecting motor control, such as ALS. Clinical trials using neurons derived from iPSCs to treat ALS are currently underway.Expanding the availability of cells for such treatments could facilitate more extensive testing and development for widespread use in humans, according to Galloway.

The research was supported by funding from the National Institute of General Medical Sciences and the National Science Foundation Graduate research Fellowship program.