This week we will talk about the transformation of human fibroblasts into hepatocytes using the biotechnological techniques: infection with lentiviral vectors and cellular immortalization. The purpose? Study a situation with a high idiosyncratic component: the damage that causes the liver of each person and the consumption of medicines. The authors? The Experimental Hepatología Unit of the Instituto de Investigación Sanitaria La Fe de Valencia… FOLLOW LEYENDO!
What does this strategy consist of?
From somatic cells (any cell in our body except sexual cells) of the individual in the studio, and transform them into cells with a hepatocyte-like phenotype (which in English is called hepatocyte-like cells) to be able to work with them in vitro form.
For him, we have to speak of the strategies: cellular immortalization and lentiviral infection; and some concept like: cell line or primary cultivation.
If we put ourselves in context, to study the liver damage of a patient the most obvious would be to show hepatocytes of the patient and cultivate them. Using cells obtained directly from an organ, the fabric is called primary culture. What is your main disadvantage? That in vitro growth is very limited. This is how it is difficult to extract hepatocytes from a patient.
Not only will you finish imagining it, the habitants will sometimes work with cell cultures. When we were able to grow cells, we were able to extend them on different “platitos” and wait for them to grow to try them out and keep them in cultivation, turning them on another plate or plate. In vitro, it is called in vitro by the original material of these plates, the glass.
How can it be an alternative to these primary crops?
Realize a transformation or cellular immortalization. To decide, to co-operate this primary culture and to induce some change so that these cells can multiply indefinitely, let us turn them to death. In this case, we would be talking about a cell line. A set of cells that you can maintain indefinitely in cultivation to work with them comfortably.
Do you disadvantages? The immortalization process could change its phenotype (the observable characteristics derived from combining genes and the environment), and they are not identical to the original cells.
How can a cell be immortalized? It may seem like a twist, but there are only so many strategies. In this case we will focus on the method followed by these researchers: using the SV40 T large antigen.
SV40 is a virus capable of infecting both humans and humans. What is interesting? A gen that houses its genome. This gene produces the protein known as the large SV40 antigen. This protein is able to block the p53 action.
We have also spoken on other occasions of this tumor suppressor, we have a small reminder. p53, known as the guardian of the genome, is the protein responsible for detecting irreparable damage in the genome of other cells and activating its mutation.
In this way, it avoids that damage in the DNA (which could end up leading to mutations and tumors, simply due to the aging of the cell) is not transmitted to new cells.
Who can get the large antigen from the SV40? One joins an essential region for the function of p53, completely blocking its action. So, this cell will be unable to detect the changes in the genome and it will never die, becoming literally and immortal.
Of course, p53 is known as a tumor suppressor because it stops the accumulation of mutations that could lead to cancer. Unfortunately, there are only one of the main genes that appear to be altered in tumor cells, which we think are also deadly.
We will now go to lentiviral infection. This concept is easily understandable if we understand how a virus works. A virus comes into contact with a cell and inserts its genetic material.
In this way, the DNA (the RNA) of the virus multiplies using our own cells, so that it can continue to infect. Well, when we refer to lentiviral infection, we are talking about cogerating these viruses, making changes in their genome so that in the future this ability to jump from cell to cell, and to change our genes or genes of interest.
How did we get there? That using a virus-friendly mechanism, we introduce into the genome of cells the genes that interest us.
And the concepts are cleared, now we can understand the work of this group of researchers: Once the patients’ fibroblasts were isolated, these cells were subjected to lentiviral infections.
Firstly, to introduce the SV40 large T antigen into its genome and immortal cells to easily work in vitro. And secondly, with a cocktail of genes needed to express a hepatocyte phenotype.
What gene cocktail? Genes for three transcription factors: HNF4A, HNF1A and FOXA3. The factors of transcription in proteins are charges to induce the expression (transformation into protein) of other genes.
These 3 transcription factors are key to expressing essential genes for a liver phenotype. In short, these transcription factors induce the expression of genes that provide a cell with the proper characteristics of a liver cell.
More details? These researchers used an inducible transcription factor expression system. Decide, these factors will only be expressed, and will transform the cell into hepatocyte, when we want it. As? Using a doxycycline-activated promoter.
Very straightforward: the sequence of any genre preceded by another sequence, the promoter. This promoter is responsible for allowing, however, the expression of the genes that they have by default. It is precisely in this region from which the transcriptional factors are united to activate the expression of the genes they control.
In this specific system, the 3 transcription factors have been inserted into the fibroblast genome with a forward promoter. Promoter that only activates and induces the expression of two factors when researchers add to the doxycycline cultivation.
If this antibiotic is not present, the cells follow the fibroblast and can be maintained indefinitely in culture. If doxycycline is used, the transcription factors are expressed and can, in turn, induce the expression of the genes that “transform” the fibroblast into hepatocyte.
The result? Cells owned by a patient (with their genome), easily isolated (from the skin), immortalized (never dying) and with characteristics of the same patient’s hepatocytes (as a result of these three genes).
Thanks to this strategy, it is possible to work in vitro with the individual cells of each patient, avoiding the use of primary cultures and their disadvantages, and I could study the characteristic changes of each patient in response to the liver by drugs.