Description of Research Work
Stem cells (SCs) are undifferentiated and self-renewal, human cells. They have ability to differentiate into a complete organism or organ or any type of tissue . They undergo mitotic division and differentiate into any type of cell, organ, and tissue or even into a complete organism. Different types of growth factors are involved in differentiation of SCs. SCs plays a very vital role in repairing, maintenance and development of different organs, cells or tissues such bones, nerves, kidney, liver, skin, muscles etc. SCs are present in human body from birth to the end of life . They are important because of their distinct characters.
SCs may be totipotent, multipotent or pluripotent. They are important for two reasons, first their self-renewing ability. Second, under proper lab conditions they can convert/differentiate into specific tissue, organ or cell having specific function. There are different types of stem cells used for clinical purposes.
Embryonic stem cells (ESCs) are pluripotent, taken at blastocyst stage. They can differentiate into any other cell type. ESCs are also used to make embryo cells for fertility treatment. Adult stem cells (ASCs) these are taken from bone marrow, muscle tissues, adipose tissues and peripheral blood . These may be multipotent, totipotent or pluripotent. ASCs are non-immunogenic and not patient specific. Examples of ASCs such as hematopoietic and non-hematopoietic SCs. Hematopoietic SCs are those that form blood cells . Another type of stem cells is induced pluripotent stem cells (iPSCs), form by reprogramming ASCs. Under which conditions and how these stem cells were formed, there is a long mechanism. By using iPSCs we can get any type of specific tissue, cell or organ. These are pluripotent stem cells. They behave like ESCs .
SCs are using to treat a number of diseases, such for treatment of diabetes, bone repair, leukemia, bone-marrow transplantation, skin grafting etc. Use of SCs technology is getting importance day by day as a regenerative medicine and providing new treatments for different diseases. Regenerative medicine is an integrative field concerned with substitution, repair or reinstatement of damaged tissue . Regenerative medicines provide cure for diseases, for which modern medicine has no treatment yet. SCs are now using also for the treatment of different skin diseases and in skin grafting, patching etc. Use of SCs in skin treatment has given a new hope to victims. It is a big breakthrough.
Skin is an important human organ, present on two different locations, in the epidermis and in the hairfolicles . Skin is also called the first line of defense against different pathogens. It also acts as immunoprotectant and as a barrier that invade the entry of any kind of pathogen . It is most important and sensitive part of human body. Damage to skin cells may also result in death of the patient. Skin damage is may be because of allergy, accident, infection, burn or acid. The increase skin damage exceeds the regenerative ability of skin, which is also a threat for other organisms and require an efficient remedial inventions. Skin regenerative ability also decreases with the age . Various therapies and techniques have been developed and many are in developing process for skin repair and for the treatment of skin related diseases. Although no exact mechanism known about skin repair.
In the past few years, various types of skin substitutes have been developed by using tissue engineering. It plays an important role in skin repair and other skin related treatments but it has limitations . Other techniques have been also used, one that is emerging is use of stem cells, which looks very promising and scientist are working on them and coming up with great ideas and inventions. Great no. of stem cells are present in skin. Working on SCs will help scientists to know more about human body and its working mechanism. Use of ESCs is not preferred because it has various ethical and social issues . The ethical issue in use of ESCs is embryo destruction. Use of ESCs also has safety issues and chance of immune rejection, so ESCs are not use much. iPSCs looks more promising, use to treat different diseases and forming specific tissues especially skin. iPSCs have provided novel ideas because using iPSCs, no chances of immune rejection and no ethical issues . They behave although like ESCs. Discovery of iPSCs technology has provided the scientist with unique tool iPSCs are now using for the treatment of skin diseases . Great no. of SCs are present in skin. Use of iPSCs opens up new way for the treatment of acid and burnt victims. We can repair, substitute or can graft the skin by using iPSCs. Use of SCs is increasing day by day which is very beneficial for humans.
So in this research we will monitor past and present clinical technologies and therapies use for skin treatment, and will provide with novel ideas by using iPSCs.
Need and Significance of the Research
We are very well aware of the healing capacity of SCs. SCs cells plays a very important role in emerging field of regenerative medicines. Day by day increasing rate of various diseases compels the researchers to find new ways to tackle the problem. In all this SCs therapies are the most novel approach the researchers come up with so far until now, in the field of regenerative medicines.
Significance of this research is, if we use human iPSCs for developing a whole new skin and transplant that skin through surgery then we will be able to treat many skin diseases. Especially the burnt victims can get their new and healthy skin back, without the risk of immune rejection because we would use patients own stem cells, which will eradicate the risk of transplant rejection.
We need this research work to be done, so that we can give new hope to the patients suffering from skin disorders and to the burnt and acid victims, to live a healthy life. Because we believe, iPSCs have ability similar to ESCs, that they can differentiate into any type of cell, they also have great potential to develop a new skin. This will be a big breakthrough in the field of surgery and transplantation.
Objective of the research
The objective of this research:
- To highlight new aspects of SCs technology by using novel approaches.
- iPSCs have a great potential to differentiate into any type of cell, so by exploiting this ability of iPSCs we decided to develop new skin of humans by using skin SCs which will form a whole new skin of that person from which SCs are taken.
- The risk of immune rejection in this case is also eliminated because patient’s own SCs will be used. So, by in this way skin transplantation of burn victims and other skin disease patients will become safer and reliable.
Review of Literature
Generation of induced pluripotent stem cells in 2006 by Shinya Yamanaka was a great revolution in the field of regenerative medicine and this discovery was made by following many different research findings which were done previously in past in the field of stem cells.
In 1954, John Enders won noble prize in the field of medicine as he grow polio virus in the embryonic cells of kidney.
In 1962, Sir John Gurdon did first cellular reprogramming. He generated tadpoles from frog’s unfertilized cells and he introduced the nucleus of intestinal epithelial somatic cells into unfertilized eggs and transplanted these eggs, he uses the nucleus of epithelial somatic cells of same frog .This method was named as somatic cell nuclear transfer (SCNT), this was the first time when reprogramming of somatic cells was done to attain the pluripotency with the alike genetic material. This discovery is supposed to be the foundation of cloning, and then after 35 years, Sir Ian Wilmut and colleagues followed the same SCNT procedure and created dolly sheep by reprogramming the somatic cells and named as somatic cloning.  These two approaches led to the conclusion that the nucleus of somatic cells which are differentiating, those nuclei possesses all that genetic information which imparts pluripotency.
In 1981, Sir Martin Evans, Matthew Kaufman and Gail R. Martin generated mouse embryonic stem cells (ESC) lines 
In 1988, Austin Smith developed suitable culture conditions to maintain the pluripotency of reprogrammed somatic cells for the long duration of time 
In 1998, James Thomson generated human embryonic stem cell (ESC) lines 
In 2001, President George W.Bush restricted the research on human derived embryonic stem cells.
In 2001, Tada and colleagues fuse somatic cells with embryonic stem cells to obtain those cells having genes which are associated with pluripotency, on the behalf of this research this point is cleared that there are some essential factors in embryonic stem cells which contribute to the pluripotency and responsible for transferring this pluripotency into the somatic cells 
In 2006, Shinya Yamanaka generated mouse induced pluripotent stem cells (iPSCs) by using OSKM cocktail (OCT 3\4, SOX 2, Klf 4, c-Myc) 
In 2007, Shinya Yamanaka generated human induced pluripotent stem cells using OSKM. James Thomson generated human induced pluripotent stem cells by using OSNL cocktail (OCT 3\4, SOX 2, Nanog, Lin 28) in the same year 
In 2009, President Barack Obama removed the restrictions that were previously put on the research related to human embryonic stem cells by George W. Bush 
In 2010, a trial was performed in which a person who got spinal cord injury become a first to be treated with human embryonic stem cells and trial was carried out under the supervision of Geron of Menlo Park, California.
In 2012, blindness is treated by using human embryonic stem cells.
In 2014, Charles vacant and Haruko Obokata and their colleagues purposed a very revolutionary concept that every cell can be converted into its pre-embryonic stage and they utilized a very simple technique that requires only 30 minutes. Masayo Takahashi carried out trial to treat blindness related to age by using induced pluripotent stem cells.
In 2015, multipotent and pluripotent stem cells were isolated from the peripheral blood of human and characterization was also done .
In 2016, a study was made to treat a lung disease called as idiopathic pulmonary fibrosis (IPF) using rat’s mesenchymal stem cells taken from adipose tissues and animal model was prepared to check the results .
In January 1, 2018, a review was published in which the research was carried out on the use of umbilical cord derived stem cells instead of bone marrow as a source of hematopoietic cells to cure a number of diseases related to blood. For example, leukemia .
Various steps are carried out to convert and an adult cell into induced pluripotent stem cells, mostly these cells are taken from a skin biopsy or from any tissue, and then by using genetic reprogramming these cells are converted into induced pluripotent stem cells (iPSCs).
Isolation of somatic cells
First, we will isolate skin cells through small skin biopsy by this way we will obtain a number of cells for reprogramming , different types of skin biopsy are being used now-a-days we will choose according to our need .
Reprogramming of skin cells
iPSCs are developed by a process called as “reprogramming” of somatic cells and this reprogramming is done by adding four transcription factors into somatic cells by using appropriate delivery system  Until now only two sets of genes are being introduced into somatic cells: OSKM (OCT 3/4, SOX2, KLF4, c-Myc) introduced into mouse somatic cells while other set of genes is OSNL (OCT 3/4 , SOX2, NANOG, Lin28) was used to create human induced pluripotent stem cell lines.
We will prefer OSNL cocktail because genetic studies shows that OCT3/4, SOX2 and Nanog play significant role in maintaining pluripotency at embryonic developmental stage. iPSCs will be differentiated unlimitedly in a cell culture having proper conditions.
We have to direct these reprogrammed somatic cells to be differentiated into epidermal keratinocytes, for this purpose we will treat the iPSCs cell culture with two components known as ; retionic acid and BMP4 ( Bone Morphogenetic Protein 4) .
Keratinocytes which will be developed from iPSCs will have capability to form epidermis layer in 3D skin equivalent culture in vitro
Identification of reprogrammed cells
To check the conversion of somatic cells that whether they are converted into iPSCs or not we will test the iPSCs colonies for the expression of genes associated with the pluripotency we can achieve this by over expressing those genes and if those cells will differentiate into three germ layers i.e. ectoderm, endoderm and mesoderm in- vivo then we can say that these somatic cells are converted into iPSCs .
Delivery of transcription factors
For delivering the transcription factors we have to choose some delivery system which will deliver those factors in somatic cells.
Non- integrating delivery systems are introduced now-a-days for safe delivery of transcription factors into somatic cells as the integrating delivery systems cause mutagenesis in host genome example of integrating delivery systems are; Retroviruses and lentiviral systems.
Non-integrating delivery systems includes; adenoviruses, Sendai virus, Episomal vectors e.t.c
We will use the improved version of Sendai virus (Sevdp) which will be easily eradicated from somatic cells and also these are very effective in delivering the genes and efficiency rate is more.
Expected Outcomes of the Research
Induced pluripotent stem cells can be divided into any cell type and in this research that ability of iPSCs will be utilized for developing a new human skin and expected outcomes of this research is as follow
- Keratinocytes can be generated from iPSCs which will form a new epidermis layer of skin.
- Moreover, along with the formation of keratinocytes from iPSCs other human skin components are also being regenerated, those components includes; melanocytes and fibroblast.
- If the three most essential components of human skin are being generated into laboratory through this research then it would become possible to regenerate a new, functional and healthy skin, those important components includes keratinocytes, fibroblast and melanocytes.