Redetermining The Fate of a Cell — From Cloning to Induced Pluripotent Stem Cells

Glossary:

Glossary:

Embryonic and Extraembryonic Cells: Embryo contains of embryonic cells that forms all cells and structures that a fetus or the baby contains whereas the extraembryonic cells are present on outside and they form structures like placenta and umbilical cord which although essential in development of a baby, is not retained in the baby.

Cell Development and Cell Differentiation: Cell differentiation is a process through which a stem cell changes its form and acquires a function. In this article, the terms cell development and cell differentiation are used interchangeably.

Pluripotent, Multipotent and Unipotent Stem Cells: Pluripotent stem cells can generate all embryonic cells but not the extraembryonic cells. Multipotent and Unipotent cells, also found in the adult body, can generate only one or few cells of one lineage.

Epigenetics: Epigenetics literally means “beyond genetics”. In this article, when we say epigenetic changes, it simply means that certain genes were switched off and others were switched on.

Enucleation: Removal of a nucleus (and hence the entire genome) from a cell.

Embryoid Bodies: These are aggregates of Pluripotent Stem Cells that are characteristics of Embryonic Stem Cells.

Aneuploid: containing abnormal number of chromosomes.

Every multicellular body starts with a single cell. This single cell, called zygote, divides to produce 2 then 4 then 8 and eventually trillions of cells of thousands of different types and different functions. Cells like zygotes and blastomeres that have the potential to give rise to cells of different types and functions are called stem cells.

Stem cells with different potentialities exist. The first cell of life — the zygote and the blastomeres are Totipotent. Meaning, they can generate all embryonic and extraembryonic cells. But as the cell differentiates its developmental (cell development) potentialities become restricted. Totipotent stem cells differentiate into pluripotent stem cells that eventually give rise to multipotent and unipotent stem cells. The last cell that a stem cell differentiates into, and which can not differentiate further is called the terminally differentiated cells and these cells are fixed in their form and function.

Consider the potentialities of a stem cell in your petri dish. One can theoretically grow a desired organ. One can study how a stem cell differentiates into an adult cell.

But, the question arises: what are the changes that transform a stem cell into a differentiated one? Do stem cells differ from adult cells genetically or do they differ epigenetically? One of the earliest SCNT (Somatic Cell Nuclear Transfer) performed by Gourdon provided the answer. Gourdon enucleated a totipotent stem cell — the zygote and transferred the nucleus of a cell from tadpoles. The modified egg still developed into a frog.

In Gurdon’s experiment, since the modified egg containing the tadpole nucleus behaved like a totipotent stem cell, it is clear that both the adult cells and the stem cells contain the same genome. Also, genetically, each cell is totipotent.

After Gurdon’s experiment, soon there was an ardent desire to establish a stem cell population in vitro that could be studied and experimented with. Teratocarcinomas were the first in queue.

TERATOCARCINOMAS

In around the 1950s, some special tumors of the gonadal region were discovered, which consisted of a haphazard mixture of several adult cell types. These were called Teratocarcinomas. When a single cell from teratocarcinomas were injected into another body, it gave rise to an entire tumor. This indicated the pluripotent stem cell characteristic of Teratocarcinomas.

Apart from having pluripotent characteristics, the teratocarcinomas contained embryoid bodies. This suggested their embryonal origin and they were eventually called the Embryonal Carcinoma Cells (ECCs).

Subsequent studies on teratocarcinomas identified unique cells with large nuclei and dark cytoplasms. These cells were the pluripotent stem cells present in the teratocarcinomas. These cells were used to establish immortal cell lines.

Pluripotent Stem cells of Teratocarcinomas were similar to Embryonic Stem Cells but there was only one difference. The differentiation patterns of ESCs are quite ordered whereas the Teratocarcinomas differentiate haphazardly.

Before we move ahead, we must define Transcription factors : A transcription factor is a protein that regulates the expression of a particular gene. In simple words, it can switch on or off a particular gene.

When pluripotent teratocarcinomas were fused with the thymocyte (somatic cell), the hybrid cells showed pluripotency. This was contradictory to the expectation as the researchers thought that factors ensuring a developed (differentiated) state of thymocyte must dominate the teratocarcinoma cells that has developmentally undetermined fate (or in other words, the teratocarcinomas must not be producing any factors and thymocyte, producing factors that ensured its thymocyte-state, should induce the thymocyte-state on teratocarcinomas and the hybrid as well). But, the pluripotent nature of the hybrid suggests that there are factors that ensure the undeveloped/undifferentiated state and that dominates over the factors that ensure the differentiated state.

But, Teratocarcinomas being aneuploid, contribute poorly to the adult somatic cells. Moreover, Teratocarcinomas induced pluripotency in somatic cells if it was fused with it. The fused hybrid, being tetraploid, can not be therapeutically used.

ESCs — THE NEXT CANDIDATE

ESCs are karyotypically normal and are derived from the Inner Cell Mass (ICM) of the embryos. These can give rise to all embryonic cell types but not the extraembryonic cells. Developmental potential of any cell can be tested using tetraploid embryo complementation and ESCs are most successful in this assay.

A tetraploid embryo can implant at higher frequency but rarely forms an embryonic structure. When aggregated with diploid embryos, the tetraploid cells are selected against, except in the extraembryonic membranes. Since the ESCs can’t generate the extraembryonic layers, when aggregated with tetraploid embryos, the ESCs form a proper embryo (with extraembryonic layer derived from the tetraploid embryo) and can generate an entire ESC-derived organism. This establishes the pluripotency of ESCs. However, the post birth viability of this ESC derived organism is close to zero.

Apart from ESCs, pluripotent stem cells have also been derived from other embryonic and adult tissues. Examples include Epiblast-Derived Stem Cells (EpiSCs), Embryonic Germ Cells (EGCs), Primordial Germ Cells (PGCs), and Multipotent Germ Line Stem Cells (mGSCs). But ESCs have been the best candidate in the tetraploid embryo complementation as they contain the balanced parental imprints (EGCs have erased imprints whereas mGSCs have only paternal imprints).

However, all of the pluripotent stem cell lines induce pluripotency in the somatic cells upon fusion. Hence, it is clear that the factors that ensure the pluripotency is dominant over the factors that ensure the state of a somatic cell.

TRANSCRIPTION FACTORS DECIDES FATE. YAMANAKA FACTORS GENERATES iPSCs

A cell adopts a fate based on its **gene expression **pattern. A cell in the eye differs from a cell in the intestine not because they contain different genes but because they express different genes. As the SCNT experiments by Gourdon and others proved, all the cells contain the same genome.

The characteristic gene expression pattern of a cell is determined by the pool of its transcription factor. So, a muscle cell has a different pool of transcription factors than a neuronal cell and hence both the cells express different genes.

But what if we supply a muscle cell, the transcription factors characteristic of a neuronal cell? The muscle cell changes its form and function and converts into a neuronal cell. This change in form and function has been experimentally done with cells of many types and almost any cell of any type can be converted into any cell of any other type, with few limitations.

But what about stem cells? There should be a pool of transcription factors in stem cells that ensures its characteristic gene expression factors. Yamanaka and Takahashi ventured to identify the crucial and master transcription factors which are characteristic of a pluripotent stem cell and which when expressed in a cell, eventually activates all other genes that together expresses the pool of transcription factors of a stem cell. This pool of transcription factors, established using the crucial or master transcription factors, then converts a cell into a pluripotent stem cell.

Although, expression of a particular transcription factor and establishment of phenotypes of a stem cell are not sequential but tend to overlap. Yamanaka and Takahashi identified four such crucial transcription factors, that were Klf4, Sox2, c-Myc and Oct4. These factors are often called Yamanaka Factors or Reprogramming Factors.

Expression of these Yamanaka Factors in a somatic cell can transform that particular cell into a stem cell. Resultant stem cells are called induced Pluripotent Stem Cells or iPSCs. A number of ways, including integrating and non-integrating viral vectors and transposons, have been used to deliver these Yamanaka Factors in the cell. The effectiveness of inducing pluripotency in the cell differs based upon the method that is used.

iPSCs have been derived from somatic cells of varied species, including humans, suggesting that the transcriptional and genetic network establishing pluripotency remain conserved during evolution.

CHALLENGES

iPSC technology, although a milestone, needs to be improved. There are several shortcomings that still pose an obstacle in utilizing this technology for medical purposes. Although very similar, iPSCs are not completely identical to ESCs.

These four Yamanaka Factors are sufficient to induce pluripotency but supplementing other factors can produce better iPSCs that are more similar to ESCs. And one or two Yamanaka Factors are replaceable with other factors.

The efficiency of generation of iPSCs is very low. When a population of cells or a cell culture is exposed to Yamanaka Factors, only about 0.1% to 1% of cells are transformed into stem cells. And the process of transformation is very slow, consuming days. Using more efficient techniques of factor delivery in the cell increases the rate and efficiency but the process is still slow and less efficient.

Two models have been used to explain the less efficient way of reprogramming somatic cells into stem cells. The first model suggests that some cells within a population have the inherent ability to transform into a stem cell. These cells are present in a very small amount in a population and only these convert into stem cells upon exposure to the reprogramming factors.

Certain cells within a population are closer to stem cells and hence this model seems reasonable. But stem cells have been derived from different cell types and hence this model can’t be true.

Another model, the stochastic model, suggests that all cells can be transformed into stem cells but they need to undergo stochastic changes. Only a few cells can do all the stochastic changes perfectly and these are converted into stem cells.

Experiments suggest an interplay of both models and the dilemma of which model is correct can be solved if we assume that cells that are closer to stem cells need very few stochastic changes, compared to other cells, to be transformed into stem cells.

THE TAKEAWAY

SCNT established that all cells, within the body, share the same genome (although different cells in a body can carry different degrees of mutation and genomes although very similar, can’t be identical) and genetically, all cells are totipotent.

Identification of Teratocarcinomas as pluripotent but haphazardly differentiating cells gave academia a new direction and people started finding other sources of stem cells and they eventually isolated Embryonic Stem Cells (ESCs).

A cell’s fate is determined by the pool of transcriptional factors that it contains which epigenetically **locks a cell into its form and function. But exposure to transcription factors from other cell types can transform a cell. This led to the identification of reprogramming factors that transform somatic cells into stem cells — the induced Pluripotent Stem Cells or iPSCs.

A lot about the cell development and differentiation was unknown before the isolation of stem cells. SCNT, teratocarcinomas and ESCs paved the way for iPSCs and these techniques and cell cultures uncovered a lot about development and differentiation. iPSC technology was a milestone but still needs to be improved. Although many therapeutics involving stem cells are already in the market, their therapeutic applications are still being explored.

References :

1. Induced pluripotency: history, mechanisms, and applications. Authors: Matthias Stadtfeld and Konrad Hochedlinger. (Review, Main Reference).

2. The Development of Transplantable Teratocarcinomas from intratesticular Grafts of Pre- and Post implantation Mouse Embryos. Authors: LEROY C. STEVENS. (Teratocarcinomas).

3. SPONTANEOUS TESTICULAR TERATOMAS IN AN INBRED STRAIN OF MICE. Authors: LEROY C. STEVENS, JR., AND C. C. LITTLE. (Teratocarcinomas).

4. Embryonic stem cells alone are able to support fetal development in the mouse. Authors: ANDRAS NAGY, ELEN GOCZA, ELIZABETH MERENTES DIAZ, VALERIE R. PRIDEAUX, ESZTER IVANYI, MERJA MARKKL’LA, and JANET ROSSANT. (ESCs and Tetraploid Developmental Assays).

5. Embryonic Stem Cell–Somatic Cell Fusion and Post fusion Enucleation Huseyin Sumer and Paul J. Verma. Authors: Huseyin Sumer and Paul J. Verma. (Cell fusion in which hybrid acquires pluripotency)

6. Fbx15 Is a Novel Target of Oct3/4 but Is Dispensable for Embryonic Stem Cell Self-Renewal and Mouse Development. Authors: Yoshimi Tokuzawa, Eiko Kaiho, Masayoshi Maruyama, Kazutoshi Takahashi, Kaoru Mitsui, Mitsuyo Maeda, Hitoshi Niwa, and Shinya Yamanaka. (Identification of factors associated with Pluripotency).

7. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors Kazutoshi. Authors: Takahashi and Shinya Yamanaka. (Induced Pluripotent Stem Cells).