Induced+Pluripotent+Cells

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Overview:
An //induced pluripotent stem cell// or iPSC is a type of stem cell derived from an adult //somatic*// cell rather than from an embryo or egg cell. iPSCs are produced by inducing the expression of specific genes in each adult cell. These genes, activated by invasive viruses, have been studied and are found to be active in all pluripotent stem cells. Scientists have therefore concluded that if the same genetic regions are "forced" to be expressed within an adult somatic cell, we can successfully rewind the internal "clock" of the cell and return it to its original, non-differentiated state.

In June of 2006, the breakthrough research on iPSCs was first revealed to the world at the International Society for Stem Cell Research conference in Toronto, Canada. Shinya Yamanaka, a quiet researcher from Japan, had been conducting studies pertaining to the induction of retrograde differentiation, the process by which an adult somatic cell is "brought back" through time to its original state of pluripotency//.// Of the 30 genes that are known to be essential in stem cell development and differentiation, Yamanaka finally isolated //four// genes that would contribute to the production of iPSCs and inserted them into a virus. This virus was able to reprogram adult somatic cells to act like pluripotent stem cells. This discovery may possibly grant scientists the key to overcoming the controversial topic of stem cell research.

A year after Yamanaka's experiment and breakthrough in 2006, James Thomson of the University of Wisconsin also completed the task of creating iPSCs in his lab using the groundwork set up by Yamanaka. However, he used an entirely different combination of four separate genes. This showed that Yamanaka's work was not just a fluke and that iPSCs could be a reality in a field of hopeful dreams and endless possibilities.


 * 1) **pluripotent** - able to differentiate into any one of the 220 cell types including skin cells, blood cells, nerve cells, etc.
 * 2) **somatic -** refers to cells of the body, rather than gametes (eggs or sperm)



Methods:
There is a plethora of ways that regular adult somatic cells can be converted into pluripotent stem cells. However, with the limitations of current technology there are only a few effective ways in which the four genes that are active in stem cells can be put into somatic cells. When iPSCs were in their infancy, a "cocktail" of four genes (//Oct4, Sox2, c-Myc, and Klf4//) was inserted into a virus. This virus would infect the host cell, place the four genes into the host DNA, and have the host cell replicate and activate these genes along with its normal genes. Types of viruses used for pluripotent induction include retroviruses and lentiviruses (a specific type of retroviruses).

However, there are problems with this method. One of the genes has been found to be //oncogenic//*. After observing iPSCs grow and differentiate, scientists found that tissues formed from the stem cells developed cancerous tumors. This is due to the foreign sequences that are introduced into the adult somatic cells. Once inserted into the adult somatic cell DNA, the foreign genes, specifically c-Myc and Klf4, are permanently inscribed into the genetic material. Therefore, c-Myc and Klf4 have the potential to cause cancer later on in the life cycle of the cell. This possibility of cancer proves to be dangerous to iPSC research as it will prevent scientists from gaining clearance for human trials and experiments.

Thankfully, scientists eventually discovered a new cocktail of genes (//Oct4, Sox2, Nanog, and Lin28//), which is not oncogenic. Unfortunately, they have also discovered that the genes were not the only sources contributing to the cancerous tumors. He disappointingly found that retroviruses used to insert the genes into adult somatic cells caused cancer as well. To solve this additional problem, scientists began using //adenoviruses// to transfer gene "cocktails" into cells. These types of viruses exhibit preferred and suitable traits that allow them to reprogram cells without causing permanent damage. However, there are several downsides to the adenovirus such as less efficiency.

There are two other methods as well that don’t involve the use of viruses. These include //plasmid transfection// and //piggyBac//. Both are currently difficult to complete with today’s technology and are inefficient in regards to cost and production.


 * 1) **oncogenic** - cancer causing

**Types of Viruses:**
//Retroviruses// make up a family of enveloped RNA viruses defined by common structures, compositions, and replicative properties. In each organism, the //pol// segment of the retroviral genetic sequence contains information for the reverse transcriptase enzyme. The reverse transcriptase enzyme, similar to transcriptase, allows retroviruses to incorporate their own genetic material into host cell DNA while also giving them the ability to replicate/multiply inside the cell. Transcriptase, an enzyme found in the cells of living organisms, is an essential component in the process of translating DNA to RNA, which ultimately leads to RNA translation that produces necessary proteins. In the case of retroviruses, reverse transcriptase works in the opposite way. Specifically, it allows the virus to take its own RNA sequence, translate/copy it into DNA which is then inserted into the host DNA. Therefore, retroviruses are able to successfully change the behavior and function of cells through reprogramming their DNA. However, there is one major fallback to the use of retroviruses in iPSC research: five of the seven genera of retroviruses are oncogenic (oncoviruses). This diverse range of viruses include anything from the feline leukemia virus to the human T-lymphotropic virus to HIV. In addition, most retroviruses cannot replicate their own DNA unless the host cell is currently dividing. Adult somatic cells take from a human are not long dividing, preventing many of the viruses from inserting the "cocktail" of genes needed for induced pluripotency.

//Lentiviruses// belong to one of the seven genera in the family //Retroviridae//. They possess many of the same characteristics as all other retroviruses, most important is the presence and utilization of reverse transcriptase. However, several other traits unique to lentiviruses make them suitable for iPSC induction. The most important: most lentiviruses are not oncogenic. They have a long //incubation period,// which is beneficial in creating iPSCs. An incubation period is the amount of time between exposure to a virus and the point at which symptoms begin to show. Lentiviral symptoms do not arise until after the iPSC process is complete. Therefore, lentiviruses can create "pure" iPSCs that do not contain harmful viral RNA/DNA. However, lentiviruses are inefficient in reprogramming adult somatic cells. Many times, scientists have found that iPSCs produced with lentiviruses had genes that were turned "off" that were necessary to cell division and genes that were turned "on" that caused improper differentiation or no differentiation at all. In simpler terms, lentiviruses have the potential to produce "dud" iPSCs. Examples of lentiviruses include HIV and FIV.

//Adenoviruses// are non-enveloped viruses composed of a double-stranded linear DNA genome. This type of virus is able to replicate its linear DNA inside mammalian cells by utilizing host cell replication and structural machinery. Once inserted into the cell, the virus is transported to the nuclear pore complex using host cell microtubules. The adenovirus disassembles and releases its DNA through the pores into the nucleus. There, the DNA is incorporated into the histones of the chromatin and effect the behavior and function of the cell just like retro and lentiviruses. Adenoviral induction of pluripotency has bee favored over retro and lentiviral methods as the viruses, such as the common cold, are not as dangerous or life-threatening. However, scientists still have not overcome the issue of permanent manipulation. The genes that these viruses put into the host cell remain in the iPSCs for the rest of the cell's life, even in the resulting tissues or differentiated cells. This can cause complications and mishaps later on in the process.

Advantages
There are several advantages to induced pluripotent stem cells. The main hope is that scientists will be able to perfect the production/synthesis of iPSCs in order to bypass the use of human egg cells and embryos. These two sources of stem cells have been extremely controversial (see @Bioethical Issues). Therefore, scientists will be able to study and develop stem cells without interference from the government or other bioethical groups.

Another advantage is that iPSCs are virtually identical to the host cells. Because induced pluripotent stem cells are derived from adult somatic cells, the genomes (DNA) are the same. This allows scientists to successfully incorporate the newly created stem cells or differentiated cells into the body of a patient without having to worry about the immune system rejecting the cells or tissues.

Disadvantages
One disadvantage to iPSCs is that the four required genes and their foreign DNA sequences permanently reside in the cells when inserted into host DNA. As mentioned before, this causes problems and complications such as "dud" iPSCs. In addition, some genes such as c-MYC and Klf4 are problematic because, in living tissue, they are linked to the development of cancerous tumors.

Goals and Potential
In the future, scientists hope to research the lineage or progression of diseases by studying how iPSCs become sick. People with diabetes and other genetic illness will benefit greatly from this type of scientific research, scientific research that has been retarded by the government and activist groups due to moral and ethical issues that accompany stem cells.

Researchers have also hoped to go even further and find ways to reprogram adult somatic cells into induced pluripotent stem cells without using any genes or genetic manipulations at all. In 2008/2009, a team of scientists accomplished an extraordinarily challenging feat by engineering and using recombinant proteins. These are proteins made from the recombination of fragments of DNA from different organisms. Instead of inserting the four genes into the adult somatic cells using viruses, scientists added the engineered proteins and experimented with cell conditions (without any genetic materials involved) until they found the exact mix that allowed them to gradually reprogram the cells back to pluripotency.

Brief Conclusive History
James Thomson, U of Wisconsin, isolates human embryonic stem cells President Bush restricts federal funding for research on human embryonic stem cells Douglas Melton of Harvard creates more than 70 embryonic-stem-cell lines using private funding and distributes free copies of the cells to researchers around the world Shinya Yamanaka, Kyoto University, turns back the clock on mouse skin cells to create the first induced pluripotent stem (iPS) cells, or stem cells made without the use of embryos. He uses only four genes, which are inserted into a skin cell's genome using retrovirus vectors Yamanaka and Thomson separately create the first human iPS cells July** Kevin Eggan at Harvard generates the first patient-specific cells from iPS cells — motor neurons from two elderly women with ALS Melton bypasses stem cells altogether and transforms a type of mouse pancreatic cell that does not produce insulin into one that does Konrad Hochedlinger at Harvard creates iPS cells in mice using the common-cold virus rather than retrovirus vectors — an important step in making the technology safer for human use Melton's team makes human iPS cells by replacing two of the four genes, known to cause cancer, with chemicals. All four must be swapped out before iPS-generated cells can be transplanted into people Yamanaka creates mouse iPS cells using safer plasmids of DNA instead of retrovirus vectors
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**References**
[] [] http://www.keck.bioimaging.wisc.edu/neuro670/reqreading/PiggyBacTranspositionReprogramsFibroblastsToInducedPluripotentStemCells.pdf [] []