In the amazing realm of biotechnology, consider picking up some normal skin cells and cajoling them to turn into specialized motor neurons the cells that make us move and manipulate our muscles. This is not the science fiction but the crux of what is called cell reprogramming, a scientific method to treat diseases that is transforming medicine. In a research paper just published by a team of researchers at MIT and Boston University as a preprint at bioRxiv, they take a root into this process. Under the direction of Nathan B. Wang and Kate E. Galloway, the team explains how the division history (proliferation) of a cell and the abundance of particular proteins, known as transcription factor, which act as the primary information givers, underlie this change. Not only does their work increase the effectiveness exponentially but also creates an opportunity to treat such conditions as ALS or spinal cord injuries.
What is Cell Reprogramming?
Cell reprogramming is a history rewrite of a cell at its simplest. Usually, body cells are specialized: skin cells (fibroblasts) defend and mend, motor neurons convey brain signal to the muscles. But scientists can beat this by adding transcription factor proteins that act like switches, turning on or off genes to direct the cell to a new identity.
Traditional procedures, such as induction of pluripotent stem cells (iPSCs), are inefficient and stochastic, i.e. unpredictable and have low yield. Direct conversion bypasses the stem cell step, and transforms one cell type directly into another. Nonetheless, it has been bedeviled by a lack of success rates that makes it difficult to study or utilize therapeutically. The authors addressed it by developing an efficient system that enhances conversion rates of one to one hundred-fold through the use of mouse and human cells as models.
Key Innovations: A Minimalist Cocktail for Maximum Impact
The team began with mouse embryonic fibroblasts and a reporter gene (Hb9::GFP) which glows green after cells develop to motor neurons enabling real time monitoring. They narrowed a cocktail of transcription factors comprising six (so called 6F) down to three key factors Ngn2, Isl1, and Lhx3 (NIL). They eliminated variability caused by several viruses by loading these into a single viral delivery system with self cleaving links.
The true game changer, however, was the introduction of a high efficiency component, a so called DDRR (having genes such as p53DD and HRASG12V) to enhance cell division). This combination reduced not only external variability such as cell to batch variability but also increased reprogramming. The yields increased 30-fold and purity (the proportion of converted cells) remained four fold higher than with older methods.
The Role of Proliferation: Why Cell Division Matters
Among the most interesting results of the study, the impact of the changed history of the cell proliferation process on the cell to have the capacity to re program is mentioned among them. The faster cell dividing ones at the early stages (hyperproliferative cells) are much more likely to succeed (up to 25 times more likely to activate the motor neuron marker and four times more likely to develop to maturity).
The researchers monitored division with dyes and separated cells into hyperproliferative and non hyperproliferative cells. Hyperproliferative ones did not just increase but mature, networked neurons. This implies that division is not only a process in which more cells are produced; it preconditions them to change, possibly by remodelling access to DNA or enhancing factor activity.
Interestingly, proliferation was differently affected by different factor cocktails. Adding specific factors (e.g. Brn2) slowed division and decreased yields whereas optimizing the order within an individual transcript (e.g. LNI: Lhx3-Ngn2-Isl1) improved outcomes, but did not change division rates.
Transcription Factor Levels: Not All Equal
In more detail, the team titrated (adjusted) concentrations of individual factors with upstream open reading frames (uORFs) and fluorescent tags to monitor them in real time. They discovered factor specific results:
Ngn2 (a pioneer factor that opens up the DNA): Greater amounts were associated with increased yields, which repeats in stem cell reprogramming.
Isl1: Isol levels did not have a great impact.
Lhx3: Had a biphasic response Lower or higher levels of reduced success were not best, although moderate levels were best.
They removed confounders by targeting hyperproliferative cells, showing that conversion is elevated by high Ngn2 and low Lhx3. They cross examined the three factors in a single cassette, and LNI combined with DDRR reached 50 percent purity with 360 percent yield, impressive in a post mitotic cell type.
Scaling up Mice to Humans: Scaling Up Therapy
Using knowledge in human skin cells of adults, which divide more slowly and produce fewer neurons, the team added proliferation enhancers such as myr-AKT, BCL2, and c-MYC. This tripled hyperproliferative cells resulting in thick webs of full grown neurons expressing markers such as the TUJ1 and MAP2.
And amazing enough, their optimized cocktail was able to work without neurotrophic factors (growth signals ordinarily required) in order to simplify protocols. Single cell RNA sequencing validated that the cells that were redesigned were comparable to natural motor neurons, although slight variations existed depending on the cocktail an indication of how factors influence maturity.
To make a practical test, they transplanted neurons of mice to brains of mice in which they were incorporated to the central nervous system. This opens the door to autologous therapies: taking a batch of skin cells to grow into motor neurons and transplant into a person to fix what is broken.
Implications and Future Horizons
The paper demystifies the black box of reprogramming as proliferation and factor levels emerge as important factors. It is a move to personalized medicine, where effective conversion could cure neurodegenerative disease without the ethical issues of stem cell.
Nevertheless, there are still problems: how to ensure safety in the long-term, how to scale to the human range, and how to tune to particular neuron subtypes. Such work has the potential to change regenerative medicine as biotechnology evolves.
Reference
Wang, N. B., et al. (2023). Proliferation history and transcription factor levels drive direct conversion. bioRxiv. https://doi.org/10.1101/2023.11.26.568736
Resources
bioRxiv Preprint Server – Access the full preprint and related studies.
MIT Galloway Lab – Learn more about the researchers’ work on synthetic biology.
Nature Review on Cell Reprogramming – A beginner’s guide to the field.