诱导多能性

Induced pluripotent stem cells, like human embryonic stem cells, can differentiate into almost any cell in the body, and therefore hold great promise in the field of regenerative medicine.

Human embryonic stem cells, or hESCs, are obtained from pre-implantation embryos, whereas fully differentiated somatic cells are used to generate induced pluripotent stem cells, which are also referred to as iPSCs.

In this video, you are going to learn about the basic principles behind generating iPSCs, a step-by-step protocol to induce pluripotency in differentiated cells, and some of the many downstream applications and modifications of this protocol.

Let’s begin by discussing the principles behind generation of iPSCs from somatic cell types.

Differentiated cells, like skin cells or neurons, are the ones whose fate is decided. They are committed to perform a particular function. On the other hand, pluripotent stem cells are the ones whose fate is undecided, and they can differentiate into any type of cell.

The process of changing the identity of an already differentiated cell to a pluripotent state is termed cellular reprogramming. This involves changing the pattern of gene expression in the cell, because the number and types of proteins produced by a cell play a major role in defining a cell’s identity.

One of the ways to induce cellular reprogramming is by inducing the expression of certain transcription factors. Transcription factors are proteins that bind to regulatory sequences within a gene. Some of these sequences are called “promoters,” and therefore promote transcription of a gene. A few transcription factors can influence the expression of numerous genes, which has a huge impact on cell identity.

The four classical transcription factors that have been demonstrated to induce pluripotency are Oct4, Sox2, cMyc, and Klf4. These factors are also known as Yamanaka factors, after the researcher who discovered their reprogramming effects.

Multiple methods can be used to induce expression of these transcription factors. The most common and efficient method is the use of a modified virus to deliver the transcription factor genes into the nucleus, where they will integrate into the genome.

In this method, the genes encoding the four Yamanaka factors are individually packaged into different retroviruses and added to differentiated cells. When the cells are exposed to modified viruses, a small fraction of differentiated cells become infected with all four transcription factor-carrying viruses. They begin to dedifferentiate until large spherical clusters of pluripotent stem cells are formed. The cluster formation helps iPSCs to create a microenvironment that is similar to in vivo stem cells, and therefore assist them in maintaining their pluripotency.

Since you now understand the basic principles behind the generation of iPSCs, let’s go through a general protocol for inducing pluripotency in mouse embryonic fibroblasts, or MEFs, using a viral transduction system.

Before starting this procedure, note that viruses can infect the cells in your body, so following safety guidelines is extremely important.

To begin the transfection process, the culture medium is removed from a plate containing a high density of MEFs, and the cells are washed with buffer solution. Next, a solution containing a protein-degrading enzyme, like trypsin, is added to lift the cells from the bottom of the dish. Culture medium is then added to the plate, and the detached cells are transferred to a centrifuge tube.

Following centrifugation, the pellet is re-suspended in the culture medium. Next, the cells are counted and the concentration is adjusted so that an optimal number of cells can be infected with virus the next day. Incubate the cells overnight.

After the cells have settled onto their new dish, old media is replaced by fresh media, and engineered viruses containing the desired transcription factors are added to the plate. The cells are then incubated with the viruses for sufficient time to allow infection to take place. After incubation, the medium containing free viruses is removed and replaced with fresh embryonic stem cell medium.

For 2-3 weeks following transformation, the cells should be grown at 37° in an incubator, and the culture media should be replaced daily.

After this time period, iPSC colonies that look similar to embryonic stem cell colonies should become large enough to be picked up. The colonies can be transferred to a fresh plate containing medium with appropriate growth factors, and allowed to grow further. In order to confirm pluripotency, a portion of the cell population is stained with pluripotency markers.

Now that you’ve seen how to generate iPSCs from differentiated cells, let’s look at some downstream applications and modifications of this highly useful method.

An important feature of iPSCs is that they can be used to generate almost any cell in the body. This example shows generation of heart muscle cells, called cardiomyocytes, from iPSCs. In order to do that, the iPSCs are transferred to non-adherent plates that allow them to form embryoid bodies, which are aggregates of pluripotent stem cells. The embryoid bodies are cultured in specialized medium containing serum and ascorbic acid, which enhances cardiac differentiation. Successful differentiation can be easily observed when some cells start to beat.

Since iPSCs can potentially differentiate into any cell type, they can also form an entire organism, like a mouse. This can be done using an assay called tetraploid complementation. First, a tetraploid embryo, an embryo containing four sets of chromosomes, is formed by fusing two cells of an early embryo together using an electric field. The tetraploid embryo is allowed to develop to the blastocyst stage. iPSCs are then injected into the blastocyst, which is then transplanted into a recipient female for gestation. The tetraploid cells are only able to form extraembryonic structures like the placenta, so animals resulting from this method are derived entirely from iPSCs.

Some researchers modify the reprogramming procedure to make the process of identifying successfully reprogrammed cells more efficient. For example, in this experiment MEFs with the ability to express green fluorescent protein under the influence of the Oct4 promoter helped researchers to easily identify cells that have acquired pluripotency.

You’ve just watched JoVE’s video on generating induced pluripotent stem cells. This video reviewed the principles behind this procedure, and a step-by-step protocol to generate iPSCs from differentiated cells. We also reviewed how this method could be applied or modified for in-lab experiments.

The discovery of iPSCs has had a huge impact on the field of stem cell biology, since it has an enormous potential for developing therapies that can be employed to treat degenerative disorders. Although much progress has been made with iPSCs, the hurdle that still needs to be crossed is the associated risk of cancer. The current reprogramming procedures have the potential to result in unregulated cell growth that may result in cancer. Therefore, more research is required to actually use iPSCs clinically. As always, thanks for watching!