The first successful isolation and generation of HESC was done in 2000 , by Thomson and coworkers , Reubinoff and coworkers (Thomson et al. , 1998 ; Reubinoff et al. , 2000). The generation and application of the stem cells have been over long time now. Initially, it started from mouse to presently the research is on the human embryonic stem cells. Human Embryonic Stem Cells (HESC) are the stem cells (SC) retrieved from the inner cell mass of the early blastocyst stage of the embryo , developing in an ART Lab. While embryos are maturing in the ART Lab , on/from day2-day5 , the embryo structure can be categorised as inner cell mass (called embryoblast) and the outer cell mass (called trophoblast) (Thompson et al. , 1998). The embryo stem cells are Pluripotent stem cells at the cleavage stage is undifferentiated , therefore , it has the potential to rise up into any differentiated cell type (Bremer S et al. , 2004).
Generation of HUMAN EMBRYONIC STEM CELLS
During the process of embryogenesis , the embryo at various stages can be used to derive the hESC. There are different mechanism to isolate and the generation of hESC for research and viable germ line such as ; Immunosurgery , Mechanical Dissection , Enzymatic Isolation , Laser Beam Isolation (Amit et al. , 2002). The embryos can be obtained at different stages to obtain ESC , deriving the hESC from the arrested embryos (day1 or day2) , the embryo at blastomere stage (day4) , the embryos at blastocyst (day5).
Derivation and Isolation of hESC Lines
Immunosurgery is the procedure of removing Zona Pellucida , for isolating the inner cell mass which is achieved by mechanical separation of the ICM from the trophectoderm . The arrested embryo (no cleavage division) are treated with Tyrode acid solution to remove the zona pellucida and further incubated in human feeder cells. The blastomeres undergo biopsy after the zona pellucida is removed. The blastocyst are used to derive hESC by isolating the inner cell mass (ICM) through immunosurgery , mechanically or culturing the whole blastocyst post removing zona pellucida (Stojkovic M et al. , 2005). The isolated ICM is provided suitable cell medium for the expansion of human embryonic cell line. The expanding cell line is regularly separated after four to seven days by enzymatic separation (enzymatic isolation) , for complete differentiation to happen (Mountford , 2008).
Culturing of hESCs lines
Once the blastomeres have been isolated , they are co-cultured in the embryonic medium. Medium essentially containing laminin and fibronectin. The presence of serum free media enhances the growth and prevent differentiation (M. Amit et al. , 2000). To avoid and prevent contamination from the animal based product media , it is reported that hESC can be cultured in human cell media called human feeder cells (A. Bongso , 1994). Besides animal and human feeder cells have limitation, a lot of factors have to be considered to culture cell line. Factors like Xeno-free environment , concentration presence of fibronectin , laminin and collagen (N. Desai et al. , 2013).
Multi-lineage Potential of cell lines
The pluripotent properties of hESCs is being able to differentiate into its lineages ; ectoderm , mesoderm and endoderms. It is important to generate cell lines from hESCs , as these derived line are used for transplantation, and are achieved by culturing the hESCs , differentiated cell types in a bioreactor under controlled in-vitro conditions (I. Roberts et al. , 2012).
Somatic Cell Nuclear Transfer (SCNT)
SCNT is a developing science technique , wherein , un-nucleated ovum of female donor is induced with nucleus of the somatic cell. This allows the cell to be capable to grow and develop under invitro conditions as a viable embryo to a blastocyst and further into a cloned organism (Wilmut et al. , 1997). The fibroblast of the host and the donor oocyte forms the SCNT-blastocyst , which further differentiates into NT-ESC’s. The SCNT derived ESC’s is used for developing hESC’s for therapeutic as well as research purpose.
In-vitro growth and maintenance
The Ideal culturing conditions for the human Embryonic Cell lines is the maintenance of physical environment which supports hESC development. Generally, hESCs grow in colonies , that must remain in a narrow size range to prevent differentiation. Therefore, the cell-cell interactions and the paracrine – autocrine signalling within the colonies has an important role in the development and in maintenance of hESC in the undifferentiated state ( (Brandenberger et al. ,2004 and Carpenter et al., 2004).
Use of Human Embryonic Stem Cells
Advancements and researches in reproductive technologies from more than two decades now , has enabled development of wide branches of medical sciences. The new era in pharmacology , nutraceuticals , biotechnology , gene development , cell based therapeutic growth and medicine. Since , the human derived stem cells have been developed, it has been appreciated worldwide for a major characteristic of pluripotent capabilities. The stem cell posses ability to regenerate into variety of cell types including cardiac and neuronal tissue, pancreatic cells, pluripotent progenitors, and also endothelial cells (Kaufman DS et al. , 2002 and Levenberg S et al. , 2002). The potential application of human derived embryonic stem cells are ; Cell Replacement Therapies have been developed for cell types like , cardiomyocytes , bone marrow cells , endothelial cells (Siu et al. , 2006) ; recent researches have been able to differentiate dopamine-producing cells for treating Parkinson’s disease (Parish , 2007 and Waese et al. , 2008). ; The ESCs differentiation to produce natural killer cells and bone tissues (Waese , 2008). ; developed and available ESCs are used in cellular screening to discover new chemical entities(NCE) which will respond to pharmacological stimuli (Jensen et al. , 2009) ; newly developed concept of modelling the genetic
disorders with embryonic cell lines by either manipulating cell lines or prenatal genetic disorder screening (Dr. Yury , 2009) ; the differentiation of the cell lines are used for dealing with Dan damages and its repair. The embryonic cell lines use strategies to repair double strand breaks because of pluripotent capabilities , which tend to posses repairing of DNA damages (Tichy ED et al. , 2010).
Problems Using hESCs
Amongst , potential application of the viable cell lines. With developing modern human sciences , it has also growing ethical and legal issues. there are factors to be considered closely which impact to the rejection of commercial use of hESCs. Evidently , the factors that contribute to rejection of hESCs are : For a viable cell line from the hESC larger number of cell production is needed which requires precise bioreactors and perfused system. ; An incomplete differentiation of the pluripotent cells of the hESCs , has the risk of forming teratoma due to residual rogue of the derived tissue. ; it is essential and crucial to practise xenofree protocols under controlled and monitored laboratory conditions , so as to ensure no risk of transmission of unwanted substances. ; the site of injection that might cause cancer , therefore , direct injection is preferred over portal vein (Bongso A , 2005).
CONCLUSION
Although ,the hESCs lines essentially helps in developing human research in live sciences biology, disease physiology , and gene functioning. The currently developed cell lines are used for drug development and its screening , tissue as well as organ transplant. The unique capabilities of hESCs to be pluripotent. The development and availability helps those patients who suffer from major diseases like :- diabetes , Parkinson , liver or cardiovascular problems.
