THERAPEUTIC CLONING

BIOLOGY AND CHEMISTRY
By: Catherine Zhang

  Introduction

 Noto and Kaga

When most people think of cloning, they think of creating an identical copy of a human being or an animal. One might think of science fiction and envision a mass army of identical soldiers. Famous examples of actual successful cloning procedures that come to mind are Dolly the Sheep and Noto and Kaga the cows. Fortunately, therapeutic cloning is very different and does not actually involve creating a physical copy of an organism. Rather, it focuses on just creating a patient-specific embryonic stem cell to be used for medical purposes. Basically, we are making stem cells.

Dolly the Sheep

 

  What is Therapeutic Cloning and how is it done?

 Therapeutic cloning is similar to cloning, except that the end result doesn’t produce a physical organism. Rather, it is more of the cloning of one’s cells. The cell that is produced is killed when its stem cells are taken away from it, so no actual organism is produced. The real goal is to create embryonic stem cells that are identical in DNA to the person needing them, using the Somatic Cell Nuclear Transfer method.
Here is how it is done.

   Firstly, somatic cells are taken from the patient. Somatic cells are adult stem cells that have a limited capability to differentiate. For example, bone marrow stem cells can only be turned into various types of blood cells.

Diagram depicting locations of somatic cells in the body

 Secondly, an unfertilized egg cell (called an oocyte) is taken from a female donor. While looking under a high-power microscope, the egg cell’s nucleus is taken out with a sharp pipette. The egg is now enucleated. The remaining empty egg is placed on a Petri dish and the nucleus is discarded.

Next, the same thing is done with the somatic cell, except this time, the empty cell is discarded and the nucleus is kept.

 Fourthly, the somatic cell’s nucleus is inserted into the empty oocyte. The newly formed cell is given several hours to adjust. This part is very crucial. Essentially, the egg cell “reprograms” the nucleus, causing it to go back into the pluripotent state, and behave the way it would if it were the actual DNA of an egg cell.

 The cell is then stimulated, usually with a shock, to start to divide. The cell will form a blacocyst, which has the inner cell mass inside.

 After several hours, and usually until there are 16 cells in the blacocyst, the stem cells are taken out, and then transferred onto a Petri dish. There they grow, and are cultured into different types of specialized cells to be injected into the patient.

Diagram illustrating the steps to therapeutic cloning

 Essentially, we just created an embryo that is capable of dividing without having to fertilize an egg with sperm. The resulting stem cells are also genetically identical to the patient who needs them, whereas a fertilized embryo only has the same DNA as its parents, and even then only half from each.  

  What is therapeutic Cloning used for?

Now you may be wondering, why go through all that trouble? What can therapeutic cloning exactly be used for? The answer is that the cells produced by therapeutic cloning can potentially cure a lot of diseases and problems inside the body. The newly formed stem cells are capable of turning into any kind of specialized cell in the body; they are pluripotent. These cells can replace any diseased, damaged or “broken” cells in the body. Theoretically, the cells could even grow new organs. This would eliminate the need for donors. For example, it could create a kidney for someone who had kidney failure. It eliminates the need to wait for an organ donor to die, or the need to cause inconvenience and discomfort to a live donor.

Image depicting the various types of tissues a stem cell could grow into

 The procedure is relatively new, but it could potentially be used to treat Parkinson’s disease, Alzheimer’s, spinal cord injuries, ALS, Multiple Sclerosis, and much more. It would be super effective for use of nerve cell related diseases since nerve cells do not regenerate on their own. Therapeutic cloning is also used to help learn more about how stem cells work and how specialized cells are developed. Scientists observe the cell as the oocyte reprograms the nucleus of the somatic cell. As well, since the DNA of the cells is identical to the patient, doctors can test specific treatment plans on the cells and see how they react. They can come up with specific treatment plans that are individualized for someone’s specific illness.

 As well, different drugs can be tested on the stem cells. There is an added benefit of testing if a certain drug would be effective on a patient before actually administering it to them, since the DNA of the cells are the same. In other words, whatever happens to the newly created cells would happen to the patient.

 Therapeutic cloning is also used to ensure that a patient will not reject the new cells that were injected for treatment. Often times, when foreign material is introduced to a patient’s body, their immune system will start to attack it if it does recognize it as their own. Essentially, when a baby is developing in the womb, their immune system has got to know every part of the body. So from the moment it is born, any new thing is basically a threat. But with the cells produced from therapeutic cloning, the DNA is identical to the receiver’s. Therefore, the body will recognize it as its own tissue and will not attack it.

Image showing the various possibilities of stem cells

  What are the drawbacks of therapeutic cloning?

 Therapeutic cloning is under attack mainly for ethical issues and the difficulty of the procedure. It is much more ethical than taking already fertilized embryos, which many argue are basically humans. However, many still argue that an embryo is an embryo – fertilized or not. Thus they say that the embryos created from this procedure could grow into humans, and that killing them would be synonymous to killing a human life. People believe that it messes with the natural order of life. There are many pro-life activists that are opposed to this cause, including many religious groups who are against the destruction of life.

A prolife activist protesting against embyronic research

 Therapeutic cloning also requires a great number of eggs in order to for the procedure to be successful. About one in 100 eggs are actually successful. Quite often the cell masses don’t make it past 8 cells before they die. There are many stability issues and success is rare. Oftentimes, mutations also occur and the cells turn into cancerous tumours. Sometimes chemicals and stimulants can be added to try to increase success rates, but that doesn’t mean that only a few eggs will only be needed. Thus many donors are needed, and it will be difficult to obtain a huge number of eggs from women. In order to have widespread use of this procedure to cure a major disease, billions of eggs would be needed. It is a great challenge that could only be over-leaped if eggs from other animals are used.

A chart showing success rates of primate cells undergoing SCNT. The important general info to highlight is the low percentage of bastocsyt formation for each type of cell. 

 What is the difference between induced pluripotent stem cells and therapeutic cloning?

  Recently, a new procedure for creating stem cells has been discovered that does not require embryonic cells. Induced pluripotent stem cells are different in that they are adult stem cells that have been reprogrammed to revert back to its pluripotent stage. Four different coding genes are added into the adult stem cells, which turn on its function to become pluripotent again instead of multipotent. One of the codes is one that can be found in cancer. It is the one that allows the cell to multiply rapidly like what occurs in the embryonic development stage. The codes include Oct-34, ones from the Sox gene family, the Klf family, the Myc family, Nanog, LIN28, and Glis1. Not all of them have to be used at once; some, like the Glis1, just help to increase the pluripotency potential of the cells. These codes are introduced using viruses introduced into the cell, so they must be carefully monitored and controlled for safety.

  Overall, many pro-life supporters see iPSCS as far more ethical than ones therapeutical cloning because they do not involve the use of human embryos or egg cells. It is more ethical since the procedure just takes cells from a patient’s body for use. The challenges are that iPSCS are far newer than the procedure for therapeutical cloning. Thus, there is far less technology available for it and it will be some time before greater advances are made. Therapeutical cloning has been around longer, so scientists are more comfortable and know its procedures, applications, and the variable factors that could affect its performances better.

Diagram illustrating the steps to creating iPSCs

 Now, the procedures I’ve outlined are very basic. In microscopic terms, there are things occurring chemically that allow these procedures to happen.

 What elements and compounds are found in the cell? 

  To understand what occurs in a cell, it is important to have an understanding of what makes up a cell. In basics, there are many simple elements found within a cell. Hydrogen, oxygen, nitrogen, carbon, sulfur, and phosphorus make up about 99% of the cell. Most of the cell is actually just water molecules, formed from hydrogen and oxygen. The basic cell elements also combine to form amino acids in our body, which are important in forming proteins and determining their functions. Different compounds also form to create nucleotides, which form nucleic acid. An example of a nucleic acid is DNA. Several different atoms form bonds to create DNA. As well, the reprogramming stage in therapeutic cloning is very important in order for the new nucleus to continue functioning to amino acids for proteins, nucleic acids, and other substances.

Diagram showing the various bonds in the DNA, made up of different elements

Diagram showing the various molecules that are formed within the cell

 

What synthetic chemicals are added to help the creation of somatic cell nuclear transfer stem cells? 

  As mentioned before, in order to increase the pluripotency of stem cells, sometimes a few chemicals are administered to the cell. One example is TSA. A small amount of TSA promoted more stable blastocyst formation, especially shown in experiments with monkeys.

Models of TSA (Trichostatin A)

 As well, interestingly enough, when the procedure is done in the presence of caffeine, it promoted the developed of SCNT embryos. That is, the formation of blastocysts were much more improved and a line of Embryonic stem cells were more successfully produced.

Models of caffeine

How can chemical reactions be used to determine if the procedure was successful and if the cells are pluripotent or not? 

  A chemical procedure can be done called fluorescence-activated cell sorting. Basically, the cells are tagged with fluorescent markers. They are special because only stem cells will become fluorescent, and normal ones will not. When sent through an electric field, the fluorescent ones will show a negative charge, separating it from the non stem cells. Thus the stem cells can effectively be collected and cultured for further development after being confirmed. 

What chemicals can be added to create induced pluripotent stem cells?

  As mentioned before, four main gene codes are added to iPSCs to revert a normal cell back into a pluripotnt one. The master genes are added using viruses. The main ones along with minor ones are the Sox gene family, the Klf family, the Myc family, Nanog, LIN28, and Glis1. Now, recently, a new discovery was made of CiPSCs. That is when you ONLY add chemicals to a cell, no gene codes or anything, to change it back to its pluripotent stage. In the past when they were testing it out, they could use some small chemical molecules to reprogram the cell, but HAD to include one gene, Oct4. Now, after careful fine tuning, a group of scientists has refined their process to no longer need Oct4. They have come up with a solution of 7 different small molecules that would revert a cell back into the pluripotent stage. Interestingly enough, through their experiments, they have found an interesting clue that may explain why amphibians can grow back whole body parts. This just shows how much there is to learn and the potential of stem cells that can be unlocked.

They even sell kits online that allow you to chemically reprogram stem cells of mice

THE FUTURE FOR STEM CELLS

  Overall, stem cells are still a very new technology. Therapeutic cloning is being replaced by new technology such as iPSCS and even CiPSCS. But right now, we have more experience with therapeutic cloning since it is the older method. Since it has been tested with more, we can have more practical applications for it. In the future, we can hope to see a world where we can completely regenerate our damaged parts through the use of stem cells. Hopefully, stem cells in the future can be reverted with a simple chemical solution. And who knows, maybe in the future, we will actually be cloning humans for real.

Maybe in the future we will have human clones!

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