Population of undifferentiated cells Essay Example

Population of undifferentiated cells Essay Background The term stem cell refers to a population of uniform cells that has the potency of turning into any specialized cell of the organic structure. These cells have two particular features. First, they must be able to self-renew. When the cell divides during cell division, one of the two girl cells produced is a root cell. This method allows for the care of the population of the root cells since some cells need to be preserved in order for the rhythm to go on. Another feature is their ability to give rise to differentiated cells. When a root cell undergoes cell division, it gives rise to two girl cells, a root cell and a precursor molecule. This precursor undergoes another unit of ammunition of division before the girl cells produced develop into a differentiated or partly differentiated cell type. Embryonic root cells are derived from the embryos. They have the possible to develop into any peculiar cell type whereas big root cells have a more limited potency. Here, the root cells from one peculiar tissue type can merely develop into another cell in the same tissue. They are by and large required to give rise to stem cells that are used to replace those that are worn out or have been damaged. The fertilization of an egg and a sperm gives rise to a fertilized ovum. This fertilized ovum contains all the familial information necessary for the development of a new person. It is said that the fertilized ovum is totipotent as every cell has the capacity of turning into a differentiated cell. The fertilized ovum so changes into a little ball of cells known as the blastodermic vessicle. The blastodermic vessicle consists of an interior mass of cells ( ICM ) which develops to go assorted cells and tissues of the immature embryo while the outer trophoblast, forms the placenta. Once the trophoblast attaches itself onto the uterine wall, gestation is established and the interior mass of cells, or embryoblast, becomes pluripotent. Pluripotency describes a province in which a cell may progress into more than one result. This period nevertheless, merely lasts for a short piece before the embryo begins to develop. Embryonic root cells are known as ES cells. Human ES cells have a normal k aryotype. They maintain high telomerase activity and perchance may let for abundant enlargement in civilization. We will write a custom essay sample on Population of undifferentiated cells specifically for you for only $16.38 $13.9/page Order now We will write a custom essay sample on Population of undifferentiated cells specifically for you FOR ONLY $16.38 $13.9/page Hire Writer We will write a custom essay sample on Population of undifferentiated cells specifically for you FOR ONLY $16.38 $13.9/page Hire Writer Stem cells are besides found in grownups excessively. Cells found in the bone marrow, tegument, intestine and respiratory piece of land invariably give new root cells, while countries such as the liver and musculus merely contribute to replace cells that have been lost through normal aging or harm. ( Bioethics briefing UOE ) There is a limited turnover of root cells in the encephalon and pancreas. ( Multilineage distinction from human embryologic root cell lines ) Evidence suggests the presence of hibernating root cells exists in some variety meats. Scientists nevertheless, claim that these hibernating cells seldom become active. Adult root cells are described as multipotent. This means that the cell may give rise to daughter cells from multiple, but still limited figure of line of descents. In many grownup cell types, root cell activity decreases as the age of the single readily additions. The Derivation of Human Embryonic Stem Cell Lines The totipotent fertilized ovum loses its ability to bring forth all the necessary tissues for nidation and fetal development when it divides leting for the formation of the pluripotent mammalian blastodermic vessicle ( Lines derived from individual blastomeres of 2 4-cell sage embryos ) . Human embryologic root cells, or helium cells, have the full potency of distinguishing into any specialized cell in the organic structure and so to look into this farther, several experiments have been carried out in the last few old ages. Surveies have been carried out in order to better civilization conditions, familial changes and distinction techniques to bring forth cells feasible for organ transplant or even drug testing. ( Derivation, growing and application ) However, the really first measure would be to bring forth uncontaminated, healthy cells. In order for successful derivation of human embryologic root cell lines, it is indispensable that there are a sufficient figure of embryos that are collected. Often the cleavage- phase embryos that have antecedently been derived by and left over from in vitro fertilisation intervention are donated with the givers consent. Majority of the cell lines that have been established have been from blastodermic vessicles between twenty-four hours 5 and 8 of their development. With the advanced engineering, these methods are being modified and assorted other techniques are besides being applied to bring forth consequences with increased success rates. The helium cells are obtained from the interior mass of cells of a blastodermic vessicle. helium cell lines were successfully derived from the ICM in 1998 for the really first clip by Thompson et Al. Five helium cell lines were derived and harmonizing to Thompson, they fulfilled his definition of a archpriest ES cell. The cells were obtained from the preimplantation embryo, the cells could go on to proliferate and stay in their uniform province and even after drawn-out civilization they were able to distinguish into all three germ beds of a human embryo viz. ectoderm, mesoderm and endoderm. ( Derivation and self-generated distinction of human embryologic root cells ) . These consequences were confirmed two old ages subsequently by Reubinoff et Al ( 2000 ) when two other ES cell lines had been established. ( Derivation, growing and applications ) . To get the cells from the ICM, the trophoblast bed of the blastodermic vessicle is removed automatically or utilizing immunosurgery and inner cell mass is so plated on mitotically inactivated mouse embryologic fibroblasts ( MEFs ) ( Derivation, growing and applications ) ( Derivation and self-generated distinction of human embryologic root cells. Amit M 2002 ) These are so cultured. Using symmetricalness division, the cells can go on to continue a stable population of root cells. They are besides capable of spliting unsymmetrically giving rise to two girl cells. While one girl cell resembles the female parent, the other can potentially distinguish into any cell of the early embryo. Using the mechanical method, the detached ICM cells either differentiate or bring forth cells that portion similar morphological features with helium cells and they test positive for alkalic phosphatase staining. They besides have normal karyotype. A popular method presents is immunosurgery. This involves the remotion of the trophectoderm cells with the assistance of antibodies that have been raised from either, BeWo cell, human serum or ruddy blood cells. The latter attack is highly dependable for the remotion of all the trophectoderm cells since in any instance if there were any cells staying, they would suppress the ICM from organizing. However an advantage the former procedure has is that it does non affect any antibodies, so the protocol remains comparatively simple and uncontaminated from animal-derived merchandises. ( Derivation, growing and application ) It has been proven that even aneuploid fertilized ovums can besides be used as a beginning for the coevals of normal helium cell lines and aneuploid helium cell lines. The latter could turn out to be utile for any types of research. Normal zygotes carry two pronuclei, one stand foring each parent. Some fertilized ovums nevertheless, that were given rise to by IVF carry one or three pronuclei. An advantage about the method that is used to obtain these cells is that they do non necessitate the mechanical method or immunosurgery. Blastocysts that were derived from aneuploid fertilized ovums frequently display an unusual morphology where it is hard to be able to separate between the ICM and the trophectoderm. Hence the method required the digestion of the zone pellucida and whole blastocycts were placed on inactivated MEFs. ( Derivation of a diploid human embryologic root cell line from a mononucleate fertilized ovum 2004 Edith Suss-Toby Human embryologic root cell lines have the potency of being used for regenerative medical specialty. For this ground, it is much preferred that a technique is used that does non necessitate the usage of animate being merchandises. About all of the experiments that have been carried out in order to develop the cell lines have used carnal merchandises. Immunosurgery requires the usage of merchandises such as anti-human anti-serum and guinea hog complement. Some claim that immunosurgery must be circumvented for future clinical tests since the animate being derived merchandises may pollute the ICMs and the cell lines that are derived from at that place. On the other manus, the mechanical method depends on the proficient accomplishments of the single transporting out the dissection, the chemical disintegration may damage the cells. Dissection by a non-contact optical maser may be the following best alternate for hatching and dissection of ICM cells from the TE cells. This attack is every bit efficient as the efficiencies suggested by the immunosurgery studies. ( Laser-assisted derivation of human embryologic root cell lines from IVF embryos after preimplantation familial diagnosing T. Turetsky 2007 ) Another method of the derivation of helium cells is from embryos that have been reconstructed utilizing the bodily cell atomic transportation techniques ( SCNT ) . This was conducted by Hwang et Al ( 2004 ) . The method requires the transportation of the karyon of a bodily cell into an enucleated giver oocyte. The cytol of this oocyte would so hush all the cistrons of the bodily cell, and activate the cistrons involved with the development of the embryo. The ICM cells would so be obtained from the cloned preimplanted embryo. ( Evidence of a Pluripotent Human Embryonic Stem Cell Line Derived from a Cloned Blastocyst Woo Suk Hwang 2004 ) Success rates in driving helium cell lines depends on the status of the blastodermic vessicles, the conditions they were isolated in, and the experience of the group. It is hard to indicate out the most successful method since some research groups publish the figure of embryos donated while others merely mention the Numberss of blastodermic vessicles used. Similarly, it is besides really likely that the published consequences are non to the full accurate. While some research groups may hold used legion embryos but were non able to bring forth any helium cell lines, others may hold successfully derived the cell lines but the consequences were non deemed successfully advanced and so consequences were non published. ( Derivation, Growth and Application ) The Growth of Undifferentiated Human Embryonic Stem Cells The civilization of ICM cells invariably requires the feeder beds. Any civilizations that take topographic point on MEFs demand to be replaced every two hebdomads so that they can go on to back up the uniform and proliferative root cells. ( Culture and Maintenance of Human Embryonic Stem Cells ) Some research workers believe that the feeder beds are non the optimal medium for the enlargement of the civilization as there is a high chance of the taint by cross-transfer of infective agents. Under feeder-free conditions, there have been important differences in the look of some cistrons and telomere length. Due to these differences, it is suggested that the media is suboptimal for the distinction of helium cell lines. ( Derivation, growing and application ) However, one time the civilized cells have been removed from the feeder bed, they are so put into a suspension civilization and the helium cells aggregate into bunchs of differentiated and uniform cells. These multicellular groups of cells are known as embryoid organic structures, EBs. The EBs advancement through a series of stairss ; between twenty-four hours 7 and 14 of station distinction development, they grow to go cavitated and cystic EBs. Human ES cells may develop into a trophoblast in civilization and besides produce a-fetoprotein and human chorionic gonadotropin into the civilization medium. The civilization survey finally would bring forth post-mitotic terminally differentiated cell types but this would depend on the conditions they were raised in. Here it is evident that helium cells in civilization represent a unrecorded theoretical account that may be used to analyze placental development. ( multilineage distinction from human embryologic root cell lines ) It is claimed that human ES cell lines have an advanced and a really consistent development. It has been observed that while the embryoid organic structures were developing, the civilizations showed grounds of different morphologies. This included catching cardiomyocytes, pigmented and non-pigmented epithelial cells and developing nervous cells. Harmonizing to Schuldiner et Al, uniform ES cell and differentiated EBs expressed receptors for legion growing factors that would consequence the development of the embryo in vivo. However, none of these growing factors allowed for distinction to one particular cell type. ( multilineage distinction from human embryologic root cell lines ) It is non merely the feeder bed that acts as a reverse for big scale production of helium cell lines, another hinderance is the clip period for the population of helium cells to duplicate. It takes about 36 hours for the figure of human embryologic cells to increase in a double sum. ( Derivation, Growth and Application ) A survey claimed that on running an array-CGH to look into for genomic rearrangements, duplicates and omissions were found in the blasotocyst-derived helium cell lines. A procedure called G-banding was so used for a elaborate observation. This was done by staining condensed chromosomes in order to detect the karyotype. It was believed that the abnormalcies were non due to the female parent s age, in fact the consequences of the experiments that were carried out strongly suggested that they were acquired in civilization. ( Human embryonic root cell lines derived from individual blastomeres of two 4-cell phase embryos Mieke Geens ) Often, the prolonged in vitro civilization, leads to high distinction rates and genomic instability. There is grounds of other cell lines exhibiting trisomy, aneuploidy and deviant X chromosomes. ( Derivation, Growth and Application ) Evidence suggests that each single helium cell line carries a alone genotype. This alone genomic sequence can be traced to the ICM of the blastodermic vessicle and it is expected that each cell line may be determined by the typical genomic discrepancies it carries. In order to see whether or non each cell line expressed signature cistrons, reflective of the discrepancies carried by each cell line, an appraisal took topographic point. helium cell lines were differentiated into the entoderm, mesoblast and exoderm and the similarities and differences of cistron look were recorded. ( Alone cistron look signatures of independently-derived human embryologic root cell lines ) It was clear that even though all helium cell lines were cultured under same conditions they still did non hold the same familial profile. When Abeyta et Al. ( 2004 ) were comparing three helium cell lines, it was concluded that 52 % of the cistrons found were expressed in all cell lines and 48 % of the cistrons was me rely expressed in one or two cell lines. ( Derivation, Growth and Application ) The initial cistron look surveies on helium cell lines suggested that the derived functions may be induced to show endoderm cistrons. This includes, pancreatic islet cistrons and insulin, somatostatin, glucagon and hepatocyte atomic factor 3 beta. It is nevertheless, still non known if the complete distinction into pancreatic cells will take to the growing of a population of big islet cells and the production of insulin. Nevertheless, the information suggests that the ES cells can trip the cistrons required for the development of all three EG beds in civilization. ( multilineage distinction from human embryologic root cell lines ) Curative Potential of Human Embryonic Stem Cells Although the survey of helium cells is still in its development phases, there is much hope for the success of the research. Theoretically, the survey can let the production of an unlimited beginning of cells that can easy distinguish into any other type of cell. They may be used to handle malignances, degenerative diseases, familial diseases, rednesss and infections and injury. helium cells can distinguish into cardiomyocytes from the embryoid organic structures. Since the attacks that have evolved have a high output from murine ES cells, the really same protocol is used in order to straight derive cardiomyocytes from helium cells. One such method was carried our by the usage of 5-aza-2deoxycytodine which enhances the distinction. The myocytes produced nevertheless, are immature and are consistent with fetal myocytes in footings of their construction and growing. This suggests that the developmental tract it that of an embryo instead than an grownup. In order for the myocytes to go clinically utilized, methods have to be able to give a bigger and purified population of cells. helium cells could potentially go a beginning for engraftable bosom cells, or other tissues. For ingrafting to be possible, one specific type of cell is required. An illustration would be in the instance of a myocardial infarction, ventricular myocytes would be required. If alternatively a sinus-nodal type of cell was added, it would turn out to be deadly. Ideally, the helium cells derived, would be transgenes free in order to take any possible method of taint that would come in the manner of the experiment. However, the transgenic tract may be used to understand the developmental tract better and this could be helpful to develop civilization conditions that would help the research without the usage of transgenes. Possibly the usage of a suicide cistron would be more utile. This manner in instance any of the grafted helium derived cells malfunction, a drug can be administered to eliminate the grafted cells. Hematopoietic root cells ( HSCs ) are the best understood cells which have been characterised and experimented with to a great extent. These cells do non back up long term grafts until late it has been discovered that cistrons that enhance hox cistrons such as hoxB4 drama a major function in advancing high blood chimerism in animate beings with grafts. While some research workers suggest the tract can do the cells to get belongingss that are characteristic to adult haematopoietic other surveies suggest the tract gives rise to a peculiar root cell erratically during cell distinction. However, surveies must be carried out sing how to reproduce these haematopoietic cells under specific conditions that will take to an efficient output. The bone marrow has been comparatively easy to analyze since HSCs have been readily accessible. Some niches nevertheless, are non accessible, that these cause major obstructions. It is indispensable that the niche of the root cells is equal for self-renewal, growing and distinction. In instances such as Fanconi s anemia, a graft could bring around the disease when the bond marrow fails due to an intrinsic defect of the hemapoietic root cells. Any disease that would destruct the root cell niche, for illustration myelofibrosis, a bone marrow graft would non be able to fixate the harm. Other conditions would include hepatic cirrhosis and Burnss. Although the mentioned conditions might be able to profit from the therapy somewhat, for maximised consequences they will hold to be accompanied with therapies that are aimed at pathophysiologic procedure of the cell that is being replaced. The development of the of the cardinal nervous system takes topographic point in early cell distinction, this is apparent as neural tissue is present in the embryoid organic structures. The neural tissue is a really specialized tissue where each type of cell has specific maps hence, it is indispensable that directed derivation is achieved. However, there are several hurdlings that must be overcome, since the derivation was non the lone debatable part. The cells must be surgically inserted into at the appropriate site. Unlike the cells of the haematopoietic system, the cells here do non come back to their right location via the blood stream. Although the synaptic connexions are fictile, the system starts organizing during fetal development. In order for efficient operation to take topographic point, the freshly synthesised cells will hold to incorporate into the bing, and to the full formed nerve cells. One hypothesis states that possibly the cells could be delivered in a precursor province which will develop into a proper niche when targeted and will turn in vivo. An advantage to this method would be that the growing of the embryologic root cells would help in the formation of the fictile connexions. The add-on of grownup nerve cells could turn out to be more complicated and integrating might non be able to be re-established. This could ensue in epileptiform and it could develop unsuitably in situ giving rise to assorted defects and tetratoma formations. A major issue is that organ transplant frequently leads to weave rejection. The immune system frequently responds to any alloantigen on the graft. This may be in the signifier of histocompatibility composites and ABO blood group antigens. In an uniform province, the root cells would show a low sum of MHC-1, nevertheless, in differentiated cells in vitro there is an addition in look by up to 2-4 crease. Although this look is much lower than that in the bodily cells, the addition can do the helium cell non to be a HLA lucifer and hence, destroyed in a mode that is dependent on T cells. One manner of get the better ofing this is by making helium cell Bankss nevertheless, a disadvantage is that the allele combination of the three MHC-1 cistrons can bring forth upto 11 million haplotypes and a diploid combinations. Possibly familial technology could be used as a method that would let for the formation of immunologically matched cell lines, this scheme nevertheless, is merely theoretical. Curative potency of embryologic root cells. Paul H. Ethical Issues Revolving Around Human Embryonic Stem Cells Most of the arguments that revolve around embryologic root cells are related to whether or non it is morally right to halt embryonic development. Those who oppose the thought believe that embryos should be allowed to develop. They regard the embryo as an person the minute the fertilized ovum is formed. Each fertilized ovum turns into a blastodermic vessicle that is implanted in the womb, and each fetus grows to go an single. They believe that from this minute onwards the fertilized ovum carries a alone DNA sequence that has neer existed before, and it neer will once more. Although, the genotype is merely one component of an person, in some instances, some advocators label this as slaying. It is true that harmonizing to science a life is formed at the blink of an eye that the egg and the sperm fuse. Yet it is besides true that we do non hold an duty towards the embryo at every phase of its development. Our duty increases as the embryo develops into an person. So, once and for all it can be said that the monetary value of an embryo is a much smaller monetary value to pay than the monetary value of an grownup person. Human embryologic root cells the German argument. Engels EM. Even though the embryo has the right to populate from the really start, those who oppose human embryologic root cell research and support the embryos claim that life does non depend on any of their cognitive or bodily characteristics . Human embryologic root cells the German argument. Engels EM Supporters believe that merely because the embryo does non hold the ability to ground or communicate is non a valid ground non to protect the embryo. They believe that merely if the female parent s wellness is under life baleful conditions, merely so can the babe be aborted and it will be ethically justified. Techniques such as SCNT are seen as condemnable offenses in states such as Germany. The transportation of familial information of one embryo into another embryo, fetus or person is considered illegal since it does non move to be in favor of the protection of the embryo. Human embryologic root cells the German argument. Engels EM With this sentiment, whether the root cells are left over from IVF or if they have specifically been created for the experiment besides does non turn out to be a valid ground for destructing an person. Although the whole intent of the survey may be to cut down the agony of many people, some believe that the embryos must non pay this necessary cost of life. Some people are surely against this practical attack. UOE bioethics briefing ) There are nevertheless, a few other grounds as to why some people are against the usage of embryologic root cells. It is claimed that if root cells are allowed to be cloned, this may take to reproductive cloning to be allowed since it leads to the devaluation of human life. Many argue, that possibly alternatively leting research on embryologic root cells, possibly experiments should be carried out utilizing big root cells. UOE bioethics briefing ) When root cells are donated, there are assorted issues that besides have to be dealt with. For illustration, the consent signifier, the handiness of givers and ownership of cell lines established are merely some of the jobs that are involved with big root cells. Apart from these issues, in order to handle patients with root cell therapy, there must at least be sufficient cells to finish the intervention. While in some instances there are no root cells for a peculiar cell or tissue, in other instances they may be highly rare. The beta pancreatic cells for illustration, research workers have still non been able to happen the root cells in the organic structure. ( Perspectives on Human Stem Cell Research KYU WON JUNG* ) The extraction of the root cells from other cells may be complicated but the proliferative potency and the sum of clip they remain active for is much less than that of the helium cells in vitro. Another major disadvantage is the deficiency of chances for the survey of embryogenesis if big root cells are used. Taking all this into history, grownup root cells do non function as a sufficient option. ( Perspectives on Human Stem Cell Research KYU WON JUNG* ) Some fear that in instance the usage of excess embryos are allowed, over clip, the populace would take this for granted and would happen the desire to O.K. of them. On the other manus, if these embryos are viewed as occasional and ineluctable, it would keep the symbolic value of an embryo in the society. Human embryologic root cells the German argument. Engels EM Alternatively, some may believe that since embryos in some topographic points are non granted the option to be used, possibly they can be imported. If ethically, the usage of embryos is allowed, it would be appropriate for the import of root cells. Equally, in topographic points where the research on root cells is purely prohibited, the import could still be allowed since the harm is already done. However, this may be an inducement for the devastation of another embryo since the blessing of importing root cells could besides be viewed as a moral blessing for the research. If the import was allowed to take topographic point in states that did non O.K. of the derivation of the root cells, there would be some ethical incompatibilities. Human embryologic root cells the German argument. Engels EM However, there are plentifulness of statements for the usage of embryologic root cells. Approximately 80 % of human embryos do non attach themselves to the uterine wall and gestation is non successful. Therefore, it can be said that although each embryo does transport a alone genotype, it is non certain that each embryo will finally go an person. Besides, most scientists consider human life to get down at the visual aspect of the crude run ; the point at which nervous development is initiated. This development takes topographic point at about twenty-four hours 14 of fertilisation even though there is no individual consensus that confirms so. ( Perspectives on Human Stem Cell Research KYU WON JUNG* ) Aboard this, it is merely after several unit of ammunitions of cell division that the cell lines are designated to either the placenta or embryo. Even after this, the fertilized ovum might split giving rise to twins but still keeping the fact that that at this phase the embryo can still non be considered as an person. In assorted beings, surveies have indicated that due to familial mosaicism, it is non really frequently that two embryos unify together giving rise to one fetus. In many instances, parents do let research to be carried out on the embryos that are left over from IVF. However, if parents do non give their consent to this, the embryos would be frozen in storage and would so be discarded old ages subsequently. ( UOE bioethics briefing ) With continued root cell research several medical jobs could be treated and even cured. Parkinson s disease, type I diabetes, spinal cord hurts, and birth defects are merely some of the conditions that can be resolved. ( http: //www.experiment-resources.com/stem-cell-pros-and-cons.html ) Frequently, faith plays a major function in 1s belief. For illustration, in 2004, when George Bush was elected president of the United Sates of America, the Protestant Christian and the Republican party opposed research on root cells. ( UOE bioethics briefing ) Claiming to support human life to those embryos that were traveling to be discarded at birthrate clinics, Bush merely allowed the usage of 21 root cell lines that had been produced before his determination. However, President Obama removed this prohibition against the research on embryologic root cells in 2009 since medical miracles do non go on merely by accident, and promised that the lost land would be made up for under his disposal. ( hypertext transfer protocol: //www.cbsnews.com/stories/2009/03/09/politics/100days/domesticissues/main4853385.shtml ) Islamic Iran is a state where the spiritual Grand Ayatollah, has highest spiritual and legislative power this is really different when compared to the other Islamic states. Most of the state s opinion and pattern is dependent on the faith. Islam describes the sanctity of the embryo in the uterus in the Quran. In 2002 nevertheless, the Grand Ayatollah declared that root cell research may go on in Iran. Saudi Arabia on the other manus perform IVFs on a regular footing, nevertheless, bring forthing embryos in order to destruct is purely prohibited so merely embryos that have been lawfully aborted can be utilised. The thought of utilizing excess IVF embryos for research is still problematic. ( Ethical Aspects of Human Embryonic Stem Cell Researchin the Islamic World: Positions and Contemplations ) Decision Human embryologic root cells have become prevalent in today s society due to their priceless belongingss. With their ability to self-renew and bring forth multilineage cell lines, these cells have the potency of transporting the name of scientific discipline to a new degree. Their ability to limitlessly provide hold made their research field a quickly developing one. Research workers are non merely working to deducing the cells but keeping them excessively. Experiments are being carried out in the hope to happen replies to assorted diseases from palsy to Alzheimer s disease. While discoursing ethical and legal issues, it is imperative that the scientific discipline that takes topographic point behind it is kept in head. As scientific discipline advancements, the ethical and legal system besides changes. Everything has its ain benefits and bounds. Given that there are rigorous guidelines overlooking all the possible issues which may do a job, all researches should be able to co-exist freely. For this a universe broad regulated system should be effectual. One that would include regulations and ordinances, adverting how many yearss old a blasto