A group of scientists from the Scripps Research Institute (TSRI, USA) has created a new method for reprogramming of adult cells into stem cells. In a study, published September 11, 2017 in Nature Biotechnology, scientists from TSRI screened a library of 100 million antibodies. As a result, they found several antibodies that could help in reprogramming skin cells into induced pluripotent stem cells (IPSC).
The creation of IPSC from more mature types cells of the body usually consists of the inclusion of genes of four transcription factors in the DNA of reprogrammed cells. The antibodies, identified by the researchers, bind to proteins on the surface of mature cells and can be used to reprogram them as a substitute for the three standard genes of transcription factors.
“This result suggests that ultimately we might be able to make IPSCs without putting anything in the cell nucleus, which potentially means that these stem cells will have fewer mutations and overall better properties,” said study senior author Kristin Baldwin, associate professor in TSRI’s department of neuroscience.
The technology of IPSC creation, discovered by Dr. Shinya Yamanaka in 2006, requires viral delivery of four transcription factor genes, Oct4, Sox2, Klf4, and c-Myc, into the nucleus of an adult cell, using lentiviral vector constructs. Co-expression of these four factors (OSKM) triggers the production of proteins that disable genetic programming of the adult cell and turns on the programming for a pluripotent stem cell.
The technology is pretty neat and allows scientists to make iPSCs from patients for the regeneration of tissues and organs, using various sources of cell production, including skin, blood and even urine. However, this method leads to the integration of the vector into a cell’s genome, which can cause unforeseen modifications of its structure and lead to the development of malignant tumors. In addition, the virus constructs themselves are antigens to humans, that is also unsafe and limits the practical applications of IPSC.
One of the problems of this procedure is that a virus insertion or overproduction of reprogramming factors may damage cell DNA, in a way that turns the cell cancerous. Another problem is that nuclear reprogramming usually yields a collection of IPSCs with variable properties.
“This variability can be a problem even when we’re using IPSCs in the laboratory for studying diseases,” – Baldwin said.
Unlike this method, during normal animal development, the cellular identity changes with the help of molecular signals that come in from outside the cell and induce changes in gene activity, without any risky DNA inclusions.
To solve this problem, the TSRI team, led by Dr. Baldwin, decided to develop an alternative non-invasive method for the creation of IPSC. Scientists have studied a huge library of antibodies – proteins that recognize and bind to specific molecules – to identify those that could replace OSKM reprogramming factors. Researchers believed that some of the antibodies, by binding to proteins on the surface of cells, could turn on the signaling cascade from the outside, which would activate the appropriate reprogramming genes from within the cell.
At the early stage of the experiment, the Baldwin’s group attempted to identify antibodies that could replace both Sox2 and c-Myc. Scientists used a large population of fibroblasts of mice, often used to obtain IPSC in experiments, and introduced the genes of two other transcription factors – Oct4 and Klf4. They then added their huge library of antibody genes to the cell population in such a way that each cell eventually contained the genes of one or more antibodies.
The researchers could then observe which of the cells began forming stem cell colonies, indicating that one of the produced antibodies had successfully replaced the functions of Sox2 and c-Myc and triggered the switch in cell identity. The DNA sequencing of these cells allowed the researchers to determine the responsible antibodies.
Thus, the TSRI team found two antibodies that can be substituted for both Sox2 and c-Myc. In similar experiments, they found two antibodies, which can replace the third transcription factor – Oct4. Scientists have demonstrated that, instead of inserting these transcription factors in the nucleus of genes, they could simply add antibodies to the culture of fibroblasts.
In this initial study, scientists could not find antibodies that would replace the function of the fourth OSKM transcription factor, Klf4. Nevertheless, Baldwin hopes that with a broader screening, she and her colleagues will eventually find antibodies that can replace Klf4.
Other research groups published various non-invasive methods for reprogramming IPSC using cocktails of chemicals, proteins or microRNAs instead of virulent gene delivery. However, the Baldwin’s study is the first use of antibodies to achieve this goal.
An additional advantage of reprogramming with antibodies is that the team was able to learn more about the signaling pathways that were naturally activated in this method.
The researchers found that one of the antibodies replacing Sox2 binds to the cell membrane with a protein called Basp1. This binding event blocks Basp1’s normal activity and thus removes the restraints on WT1, a transcription factor that works in the cell nucleus. WT1, unleashed, then alters the activity of multiple genes, ultimately including Sox2, to convert cells into stem cells using a different order of events than when using the original reprogramming factors.
IPSCs obtained by reprogramming using antibodies can solve some of the long-standing problems associated with traditional reprogramming techniques and can provide additional information on the complex signaling that is required to turn an adult cell into a pluripotent state.
Baldwin and her team are currently engaged in the search for antibodies that will reprogram human cells (rather than mice) in IPSCs.