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Regenerative Medicine – a Paradigm Shift from ‘stem Cells’ to ‘transdifferentiated Cells’

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Introduction

The human capacity to regenerate cells and tissues is limited. Diseases and injury often result in degeneration and loss of cells with devastating consequences. Regenerative medicine aims at replenishing such cells. We live in an era where scientists can grow the majority of cell types in their laboratories, the nerve cells, the heart cells, the liver cells, the pancreatic cells, almost any cell that you want!! One of the major aspects of regeneration is the phenomenon of ‘transdifferentiation’. Transdifferentiation (TD) is the conversion of mature, differentiated cell types performing a specialized function into another. (see BOX-1). TD can occur within a cell lineage or across lineages. TD is basically reprogramming of adult cells as they retain their plasticity and it is in contrast to our long-held view of differentiation being irreversible. Actually, the phenomenon of TD as an option for cellular reprogramming has been known for several decades; however, its application in regenerative medicine has recently gained a global burst of enthusiasm. We review here the paradigm shift in regenerative medicine from using ‘stem cells to ‘TD’, and the potential use of TD that has compelled the scientific community to show fresh interest in cellular reprogramming.

Regenerative Capacity in Stem Cells

The ability of a cell to acquire its functional state during the formation of an embryo is broadly called ‘differentiation and its ability to form the various cell types defines itfertilized’. A fertilised egg can be considered a totipotent stem cell as it has the capacity to form all cell types of the body. As lineage restriction sets in, embryonic stem cells are known as pluripotent stem cells are formed and gave rise to multipotent stem cells that are retained through adult life and are called adult stem cells. For example, bone marrow stem cells can give rise to various blood cell types. Although ‘bone marrow stem cells are the most important and abundant adult stem cells, we also have tiny amounts of other multipotent ‘tissue-specific adult stem cells’ in virtually every organ of our body. For example, neural stem cells in the brain, cardiac stem cells in the heart, satellite cells in skeletal muscle so on and so forth. These are extremely few in number and very difficult to isolate. They reside in our tissues in a quiescent (dormant) state and are activated for wound healing in case of tissue injury or disease. Owing to their presence one would assume that they can help us regenerate an entire organ in vivo, but this is not true simply because wound healing and regeneration are different phenomena with varying mechanisms, and unfortunately, we possess a very limited regenerative capacity.

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How differentiation/regenerative capacity is retained in adult cells has been of basic and clinical interest for a long time. Earlier it was thought that as the cell loses its potency when traveling from totipotency to a terminally differentiated cell and the nucleus undergoes irreversible changes in a unidirectional manner. Some scientists challenged this idea and argued that the nucleus of a specialized cell retains its totipotency, but in a dormant state; if put in the right environment, the nucleus of a specialized cell can help the development of an entire organism. In the 1950s, Briggs and King transplanted a nucleus from a frog embryo in the blastula stage of development into a fertilized egg whose own nucleus had been removed. Interestingly, these transplanted eggs developed into tadpoles. However, their results were inconclusive, and critics argued that unceasing nuclear totipotency would be accepted only if the donor nucleus was from a specialized or somatic cell. In the 1960s, John B. Gurdon used the nucleus from a specialized cell (skin cells) of a frog and transplanted it into an enucleated egg and to his amazement, embryonic development continued normally and the tadpole stage was reached. These experiments gave the first proof that differentiated adult cells retained their potency and could be possible candidates for manipulation for regeneration.

Just as investigations were carried out in adult cells, studies were also being carried out on embryonic stem cells. An example of embryonic stem cells studied extensively were mice teratomas which had characteristics similar to cells in the blastocyst. This fascinated scientists as it was soon realized that embryonic stem cells can be used to introduce germline changes for studying developmental biology and/or used as a source for differentiation of nearly all cell types towards a therapeutic end. Much later, in 1981, Evans, Kaufman, and Martin made the first in vitro ‘mouse’ ES cell line. Subsequently, germline mutations could be introduced into mice and knock-out mice were created. Evans, Capecchi, and Smithies were awarded the 2007 Nobel Prize in Physiology or Medicine for their work on embryonic stem cells. In 1998, Thompson and colleagues made the first in vitro ‘human’ ES cell line. This was accomplished using left-over embryos from in vitro fertilization procedures.

The development of a human embryonic stem cell line was a boost to the stem cell community as it provided an abundant supply of human ES cells. However, ES cells posed several technical challenges from a therapeutic perspective. Firstly, they were loaded with ethical issues, secondly, they carried a risk for rejection of the donor’s cells by the recipient’s immune system, and thirdly, they posed an inexcusable danger of spontaneous tumor formation, making them unsuitable for therapeutic use in regenerative medicine.

It was slowly appreciated that cells could be reprogrammed. This surely involves molecular manipulation at the genomic level. Of all the molecules that modulate gene expression transcription factors are known to be the ultimate players interacting with the DNA molecule. Yamanaka and colleagues took mouse fibroblast cells and forced them to increase the expression of four key genes by adding their respective transcription factors Oct4, Sox2, Klf4, and Myc. The expression of these genes, he knew, are usually lost somewhere along with development, and their re-introduction takes the cells back into their origins i.e. reversing the cell’s potency. And voila, they reverted or de-differentiated into their pluripotent state just like embryonic stem cells! These factors came to be known as ‘Yamanaka factors’ and he called these cells ‘induced pluripotent stem cells or iPSCs. Once pluripotent, they now had the ability to be transformed into any cell type of the body using a different cocktail of chemicals. It was brilliant because this meant that skin cells could be taken from a Parkinson’s patient and turned into an undifferentiated or pluripotent state which could then be coaxed into becoming dopaminergic neurons and put back into that patient. In 2007, he demonstrated the same results using human fibroblast cells. Shinya Yamanaka and John B Gurdon shared the 2012 Nobel Prize in Physiology and Medicine for their respective work.

Though free from ethical issues and immune rejection, it was soon discovered that iPSCs had their own set of problems. Since reverting to the pluripotent state is not an easy task, iPSCs were technically challenging and time-consuming to make, expensive. Moreover, the risk of spontaneous tumor formation as seen in ES cells persisted even with iPSCs.

Transdifferentiation (TD)

Scientists began to speculate that there had to be a better way to transform one cell type into another. Then came a new kid on the block – ‘transdifferentiation (TD)’! As stated earlier, TD is the differentiation of one mature cell type into another without the intermediate state of pluripotency. This phenomenon was first introduced by Selman and Kafatos in the 1970s. They observed that during ‘metamorphosis’ in the silk moth, cuticle-producing cells transdifferentiate into salt secreting cells. Subsequently, it was found the surgical removal of the lens from a newt’s eye provokes the pigmented epithelial cells of the iris to transdifferentiate into lens fiber. Recently, it was also found that rectal cells transdifferentiate into neurons in the worm C elegans, and this is absurdly a part of its normal developmental process. While are all naturally occurring examples during metamorphosis/development or due to injury/surgery, nobody thought that it was possible to actually transdifferentiate cells in the laboratory. In 1987, a landmark study by Davis and colleagues reported an in vitro experiment which resulted in the TD of one adult cell type into another. As we know, all cells in our body have the same DNA owing to genome equivalence, the only thing that makes cell types different is the difference in gene expression which is in turn dependent on different ‘transcription factors. Davis and colleagues introduced a transcription factor called MyoD into fibroblast cells using a technique called ‘cDNA transfection’ (see Box-1). To their amazement, their experiment resulted in multinucleated muscle cells (instead of fibroblasts). The identity shift from fibroblast to muscle was because MyoD is a ‘master gene regulator’; simply put, a transcription factor that regulates other transcription factors making it in charge of a cell’s identity. Thus, by introducing the cDNA of a gene in ‘charge’ of a particular cell type the desired cells were obtained. Although this work didn’t gain much attention when it was reported, it certainly laid the groundwork for future experiments in this area. Despite TD being known, stem cell scientists took long to accept its occurrence in vitro believing that there may be cell culture artifacts. Additionally, Dr. Yamanaka’s work on iPSCs inspired the scientific community to dig deeper in this area and begin investigating the behavior of cells from a regenerative perspective. It took a few decades for a surge of excitement wherein many researchers delved into this subject and we started hearing of in vitro conversions of lymphocytes into macrophages, exocrine into endocrine pancreatic cells, fibroblasts into neurons, fibroblasts into cardiomyocytes, and many more. Even so, the entire range of conversions of different cell types is yet to be seen

Transdifferentiation in Humans

Bizarrely, TD sometimes occurs inside us humans. While this is true, there are very few known examples of TD in vivo (whether human or any other animal) as compared to in vitro. The most famous example of human in vivo TD is between two types of pancreatic cells. On performing pancreatic ablation, the alpha cells in the pancreas (which secrete glucagon) can spontaneously become beta cells (which secrete insulin), the latter being an important functional entity of the pancreas. Such spontaneous conversion is also seen in the case of diabetes mellitus. Therefore, a major focus of diabetes research is on transdifferentiated beta cells from a therapeutic perspective. Intriguingly, it was also found that pancreatic cells can transdifferentiate into hepatocytes (liver cells) and this is probably because they stem from common ‘progenitor cells’ during development. Again, a master gene regulator switch is enough to cause this conversion and it could have applications in treating liver diseases. Notwithstanding, between the two organs, the liver does have a limited regenerative capacity (called compensatory regeneration) which is not the case for the pancreas. Hence, of late, researchers are exploring the possibility of converting hepatocytes into pancreatic beta cells. While this has brought a new ray of hope for diabetes; it still needs to be translated from bench to bedside. Also, it should be noted that spontaneous transdifferentiation in vivo in adults (and not during embryo development) is usually caused by injury/disease and occurs between cell types that arise from a common lineage. An exception to this is epithelial to mesenchymal transition (known as EMT) which occurs as a part of normal embryonic development, besides also being a core process involved in metastatic cancers.

Mechanisms That Govern Transdifferentiation

It is quite certain that any change in a cell’s identity whether it may be differentiation, differentiation, or transdifferentiation, all involve a hallmark change in gene expression. This means a set of genes are activated and another set of genes are suppressed. During differentiation, genes related to cell proliferation are suppressed and genes related to differentiation and the new phenotype are activated. Often, a change in a master gene regulator (a gene whose protein product is a transcription factor that regulates other genes) or a change in a couple of important transcription factors (that regulate the expression of important genes) is sufficient to cause a change in the cell’s identity. Even though transcription factors are at the core of the process of TD, much emphasis has also been laid on epigenetics. A close cousin of genetics, epigenetics does not deal with alteration in the genetic sequence itself, but modification in the way genes are expressed leading to a different phenotype. These modifications majorly include ‘DNA methylation’ and ‘histone acetylation leading to chromatin remodeling. As a result, the chromatin of important genes may be loosened or tightened, thereby changing its possibility of being expressed. Further, these modifications are heritable or can be new, and can be triggered by the outside environment. In congruence, it has also been shown that changing the extracellular environment in vitro can trigger a series of changes that can lead to TD. The term epigenetics was coined by Waddington in 1942 who also introduced the concept of ‘epigenetic landscape’. This, he explained, was analogous to marbles rolling down a hill, our stem cells lose their stemness or potential and become more lineage-restricted during differentiation. Hence, dedifferentiation into pluripotent stem cells and redifferentiation into a new cell type, as seen in iPSCs, involves rolling back up the hill before making another downward route. However, TD can occur directly without a complete de-differentiation saving time, energy, and effort.

Conclusion

Although TD is direct reprogramming from one specialized cell to another, it does technically involve a step of transient de-differentiation. However, the level of de-differentiation may not be going back all the way to pluripotency. This ‘so-called’ intermediate state does not last for long because no factors are provided to maintain this state. Hence, in the case of TD, there is a lower risk of spontaneous tumor formation which is a major advantage for its application in regenerative medicine. Even so, it would be cells misleading to state which is a better option in the context of regenerative medicine. One major challenge with the use of TD is its reproducibility in vitro. Presently, our knowledge of TD is still in its infancy and a lot of effort needs to be steered towards it to tap its genuine potential.

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