Stem Cells: New Trend in Therapeutics

Stem Cell (SC) is a magic seed as organism develops from a single cell called Embryonic Stem Cell (ESC). In adults, healthy cells from Adult Stem Cells (ASC) can replace damaged cells. ESC are capable of developing into any of 220 cell types that make up the human body. Because ESC are the precursor cells to all other cell types in the human body, this accomplishment has set the stage for a revolution in medicine and basic biology. This article reviews information about SCs and its therapeutic applications.

1. Introduction:

SC is an undifferentiated somatic cell that is capable of either division to give rise to daughter SC or differentiating into any specialised cell type^1,2. These cells are capable of dividing for indefinite periods in culture to give rise to specialised cells. These cells are called pleuripotent cells, as they are capable of giving rise to most of the tissues of an organism. Human development begins when a sperm fertilizes an egg and creates an embryo. Taken from human embryo only 5-7days old SCs are capable of developing into any cell types that make up the human body ¹.They have the ability to replicate indefinitely and morph into any kind of specialised cells by a process called differentiation².

Scientist discovered ways to obtain or derive SCs from early mouse embryo more than 20 years ago³. In November 1998, a group of scientist led by University of Wisconsin-Madison, developmental biologist James Thomson became the first in the world to successfully isolate and culture human ESC³.

ESCs are of great interest to medicine and science because of their ability to develop into virtually any other cell made by the human body. If SCs can be grown and their development directed in culture, it would be possible to grow cells of medical importance such as bone marrow, neural tissue or muscle.

2. Classification of Stem Cell ²:

SCs are classified into following different types.

1. Pluripotent: Pluripotent SCs can turn into several other type of cell, but not into anything.

2. Totipotent: Totipotent SCs can be turned into any other type of cell at all.

3. Embryonic: Embryos contain many types of Stem Cells that are probably rare or absent in adult. They have different biochemical characteristics. ESCs are usually found in the ectoderm (skin, nerve, and brain), mesoderm (muscle, bone) and endoderm (liver, gut).

4. Tissue Specific (Adult): Many tissues in the adults able to renew themselves if damaged through growth of new cells from SCs within the tissue like bone marrow and lining of the gut. However, because ASCs are already specialised, their potential to regenerate damaged tissue is very limited i.e. skin cells will only become skin and cartilage cells will only become cartilage.

3. Sources of Stem Cells:

SCs are found in human embryos, umbilical cords and placentas⁴. Human embryos must be destroyed to retrieve SCs. Scientist can obtain these embryos from different sources, each with its own ethical challenges. First source is from fertility clinics^5,6. In fertility clinic, embryos were created for infertility purposes through in-vitro fertilization procedures and when they were no longer needed for that purpose, they were donated for research with informed consent of donor. Second source is abortion clinic. The third source is cloning: few companies have considered selling its SCs derived from cloned human embryos to other researchers. Fourth source is made to order, SCs can be made available by mixing sperm and eggs expressly to create embryo. Recently two advances in research suggest that adipose tissues (fat) could soon be used as an unlimited abundant and ethically sound source of SCs for use in transplants and cell based therapies⁷.

4. Characteristics of Stem Cells:

SCs have two important characteristics that distinguish them from other cells. First, they are unspecialised cells that renew themselves for long periods through cell division. SCs have ability to divide continually to form new types of cells. During embryonic development, even more potent SC exist that can generate many different types of tissues in the human body⁸. The second is that under certain physiologic or experimental conditions, they can be induced to become cells with special functions.

One of the fundamental properties of a SC is that it does not have any tissue specific structures that allow it to perform specialised functions. A stem cells cannot work with its neighbours to pump blood through the body (like a heart cell); it cannot carry molecules of oxygen through the bloodstream (like a red blood cell); and it cannot fire electrochemical signals to other cells that allow the body to move or speak (like a nerve cell); however, unspecialised SCs can give rise to specialised cells, including heart muscle cells, blood cells or nerve cells.

Unlike muscle cells, blood cells, or nerve cells - which do not normally replicate themselves - SCs may replicate many times. When cells replicate themselves many times over it is called proliferation. A starting population of SCs that proliferates for many months in the laboratory can yield millions of cells. If the resulting cells continue to be unspecialized, like the parent SCs, the cells are said to be capable of long-term self-renewal. The specific factors and conditions that allow SCs to remain unspecialised are of great interest to scientists.

5. Embryonic Stem Cells:

ESCs, as their name suggests, are derived from embryos. Specifically, ESCs are derived from embryos that develop from eggs that have been fertilised in-vitro in an in-vitro fertilisation clinic - and then donated for research purposes with informed consent of the donors⁶. They are not derived from eggs fertilised in a woman’s body. The embryos from which human ESCs are derived are typically four or five days old and are a hollow microscopic ball of cells called the blastocyst. ESCs are undifferentiated cells that are unlike any specific adult cell. However, they have the ability to form any adult cell. Because undifferentiated ESCs can proliferate indefinitely in culture, they could potentially provide an unlimited source of specific, clinically important adult cells such as bone, muscle, liver or blood cells⁹.

Growing the Stem Cells in laboratory:

In the 3 to 5 day old embryo called a blastocyst, a small group of about 30 cells called the inner cell mass gives rise to the hundreds of highly specialized cells needed to make up an adult organism.. In the developing fetus, SCs in developing tissues give rise to the multiple specialized cell types that make up the heart, lung, skin, and other tissues.

Growing cells in the laboratory is known as cell culture¹⁰. Transferring the inner cell mass into a plastic laboratory culture dish that contains a nutrient broth known as culture medium isolates human ESC. The cells divide and spread over the surface of the dish. The inner surface of the culture dish is typically coated with mouse embryonic skin cells that have been treated so they will not divide. This coating layer of cells is called a feeder layer. The reason for having the mouse cells in the bottom of the culture dish is to give the inner cell mass cell a sticky surface to which they can attach. In addition, the feeder cells release nutrients into the culture medium. Recently, scientists have begun to devise ways of growing ESCs without the mouse feeder cells. This is a significant scientific advancement because of the risk that viruses or other macromolecules in the mouse cells may be transmitted to the human cells.

Over the course of several days, the cells of the inner cell mass proliferate and begin to crowd the culture dish. When this occurs, they are removed gently and plated into several fresh culture dishes. The process of replating the cells is repeated many times and for many months, and is called sub-culturing. Each cycle of sub-culturing the cells is referred to as a passage. After six months or more, the original 30 cells of the inner cell mass yield millions of ESCs. ESCs that have proliferated in cell culture for six or more months without differentiating, are pluripotent^11,12, and appear genetically normal, are referred to as an ESC Line. Once Cell Lines are established, or even before that stage, batches of them can be frozen and shipped to other laboratories for further culture and experimentation.

Stem Cell differentiation:

The specific factors and conditions that allow SCs to remain unspecialised are of great interest to scientists. It took 20 years to learn how to grow human ESCs in the laboratory following the development of conditions for growing mouse SCs. Therefore, an important area of research is understanding the signals in a mature organism that cause a SC population to proliferate and remain unspecialised until the cells are needed for repair of a specific tissue. Such information is critical for scientists to be able to grow large numbers of unspecialised SCs in the laboratory for further experimentation.

As long as the embryonic SCs in culture are grown under certain conditions, they can remain undifferentiated (unspecialised). However, if cells are allowed to clump together to form embryoid bodies, they begin to differentiate spontaneously. They can form muscle cells, nerve cells, and many other cell types. Although spontaneous differentiation¹³ is a good indication that a culture of ESC is healthy, it is not an efficient way to produce cultures of specific cell types. Therefore, to generate cultures of specific types of differentiated cells - heart muscle cells, blood cells, or nerve cells, for example - scientists try to control the differentiation of ESCs. To keep the SCs growing in culture without becoming differentiated requires some very specific growth conditions and SC growth factor which are different for different types of cells².They change the chemical composition of the culture medium, alter the surface of the culture dish, or modify the cells by inserting specific genes.

Signals that regulate the proliferation of stem cells:

Scientists are just beginning to understand the signals inside and outside cells that trigger SC differentiation. The internal signals are controlled by a cell's genes, which are interspersed across long strands of DNA, and carry coded instructions for all the structures and functions of a cell. The external signals for cell differentiation include chemicals secreted by other cells, physical contact with neighboring cells, and certain molecules in the microenvironment.

6. Adult Stem Cell:

An ASC is an undifferentiated cell found among differentiated cells in a tissue or organ, can renew itself, and can differentiate to yield the major specialized cell types of the tissue or organ. Differentiation of ASCs is shown in Table 1. The primary roles of adult SCs in a living organism are to maintain and repair the tissue in which they are found. Some scientists now use the term Somatic SC instead of ASC. Unlike ESCs, which are defined by their origin (the inner cell mass of the blastocyst), the origin of ASCs in mature tissues is unknown. Research on ASCs has recently generated a great deal of excitement. Scientists have found ASCs in many more tissues than they once thought possible have. This finding has led scientists to ask whether ASCs could be used for transplants. In fact, adult blood forming SCs from bone marrow have been used in transplants for 30 years. Certain kinds of ASCs seem to have the ability to differentiate into a number of different cell types, given the right conditions. If this differentiation of ASCs can be controlled in the laboratory, these cells may become the basis of therapies for many serious common diseases.

ASCs typically generate cell type of the tissue in which they reside¹⁴. The history of research on ASCs began about 45 years ago. In the 1960s, researchers discovered that the bone marrow contains at least two kinds of SCs. One population, called Hematopoietic SCs, forms all the types of blood cells in the body. A second population, called bone marrow Stromal Cells was discovered a few years later. Stromal cells are a mixed cell population that generates bone, cartilage, fat, and fibrous connective tissue¹⁵.

ASCs have been identified in many organs and tissues. One important point to understand about ASC is that there are very small numbers of SCs in each tissue. They are thought to reside in a specific area of each tissue where they may remain quiescent (non-dividing) for many years until they are activated by disease or tissue injury. The adult tissues reported to contain SCs include brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin and liver¹⁶.

Every 60 seconds, a human being must generate about 120 million granulocyte and 150 million erythrocyte¹⁷, as well as numerous mononuclear cells and platelets from SC of red bone marrow. In the adult, SCs of the red bone marrow are able to generate several different cell lines: red blood cells, several types of White Blood Cells and Platelets¹⁸. The differentiation of mammalian RBC in bone marrow (a process called erythropoises) is one of the most striking examples of specialization occurring in nature¹⁶.The cells responsible for this remarkable productivity are derived from a relatively small number of self renewing, Pluripotent SCs laid down during embryogenesis¹⁹. In adulthood, only SCs that produce the different Blood Cell Lines remain

Table 1. Differentiation of Adult Stem Cells.

7. Therapeutic applications of Stem Cell:

SC biology has a great potential in saving many lives. SC research lies on embryo cells or SCs because they can grow into all types of cells & tissues in the body. Research with ESCs not only helps us understand the basic mechanisms of tissue differentiation but it also holds great promise for cell therapy, the use of special cells to treat disease20. It can cure many diseases like Parkinson’s and Alzheimer’s21 disease, osteoarthritis, rheumatoid arthritis, malignancies, inborn errors of metabolism and many more22. Using SC therapy, organ donations could be reduced in favour of introducing new cells grown in laboratories instead of new organs from living or recently deceased donors9. The important applications of SCs are listed below.

Bone marrow transplantation²³:

Now a days peripheral blood SC transplantation is recommended than bone marrow transplantation to rescue the patient from potentially lethal effects on bone marrow of ablative therapy given in an attempt to eradicate all traces of disease. In this process Peripheral Blood Progenerator Cells (PBPC) are transplanted rather than bone marrow. Initially PBPC circulate in peripheral blood in low concentration after the treatment of Granulocyte Colony Stimulating Factor (G-CSF) the number of circulating SCs increases 10 time.

Parkinson’s Disease (PD):

PD is a very common neurodegenerative disorder that affects more than 2% of the population over 65 years of age. PD is caused by a progressive degeneration and loss of dopamine (DA)-producing neurons, which leads to tremor, rigidity, and hypokinesia (abnormally decreased mobility). Factors that support this notion include the knowledge of the specific cell type (DA neurons) needed to relieve the symptoms of the disease. In addition, several laboratories have been successful in developing methods to induce ESCs to differentiate into cells with many of the functions of DA neurons. In a recent study, scientists directed mouse ESCs to differentiate into DA neurons by introducing the gene Nurr1. When transplanted into the brains of a rat model of PD^24,25, these SC-derived DA neurons reinnervated the brains of the rat Parkinson model²⁶, released dopamine and improved motor function. Regarding human SC therapy, scientists are developing a number of strategies for producing dopamine neurons from human SCs in the laboratory for transplantation into humans with Parkinson's disease. It is thought that PD may be the first disease to be amenable to treatment using SC transplantation^27,28.

Heart disease²⁹:

Transplanting healthy heart muscle cells can provide new hopes for patients who are suffering from heart disease, whose hearts can no longer pump adequately. SC studies have raised the hope to develop heart muscle cells from human SCs and transplant them into failing heart muscle in order to augment the function of failing heart.

Type 1 Diabetes Mellitus (DM)³⁰:

Another important disease is Type 1 diabetes, where production of insulin by specialized pancreatic cells called islet cells is disrupted. Studies suggest that transplantation of either the entire pancreas or isolated islet could replace the need for insulin injections. Islets Cell Lines derived from SCs can be used for diabetes research and eventually for transplantation.

HIV & Cancer:

The research on Embryonic and Adult SC is going on, which may lead to better treatment of HIV & Cancer³¹. An unusually intensive assault on the cancer multiple myeloma using two rounds of high dose chemotherapy followed each time by a SC transplant appears to double patients’ long-term chances of survival³². SCs from bone marrow have proven far more effective at delivering therapeutic genes to tumors, promising a safer gene therapy for cancer treatment³³.

Engineered Stem Cells^34,35:

Gene therapy for the treatment of disorders involving blood cells can be cured by ESCs. Hematopoietic SCs are removed from an affected individual and transfected with functional genes. The Engineered SCs are then reinjected into the individual.

Chimera³⁶:

Chimera is an animal, which is a mixture of several other animals. Chimeras can be made by mixing totipotent SCs with the early embryo. They can be incorporated into that early embryo, the resulting mouse has cells derived from ESCs in many tissues.

Drug discovery³⁷:

The first potential applications of human ESC technology may be in the area of drug discovery. The ability to grow pure populations of specific cell types offers a proving ground for chemical compounds that may have medical importance. Treating specific cell types with chemicals and measuring their response offers a short-cut to sort out chemicals that can be used to treat the diseases³¹ that involve those specific cell types. SC technology, therefore, would permit the rapid screening of hundreds of thousands of chemicals that must now be tested through much more time-consuming processes.

Study of Human Development:

The study of human development also benefits from embryonic SC research. The earliest stages of human development have been difficult or impossible to study. Human ESCs offer insights into developmental events that cannot be studied directly in humans in utero or fully understood with animal models. Understanding the events that occur at the first stages of development has potential clinical significance for preventing or treating birth defects, infertility and pregnancy loss¹⁴. A thorough knowledge of normal development could ultimately allow the prevention or treatment of abnormal human development. For instance, screening drugs by testing them on cultured human embryonic SCs could help reduce the risk of drug-related birth defects.

8. Conclusion:

Experiments using human ESCs for research considered to be important, to improve scientific knowledge in basic research, or to improve medical knowledge of the development of diagnostic, preventive or therapeutic processes for the treatments of human.

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Mr. Bumrela S. B*.:

Completed M. Pharm in Biopharmaceutics from Govt. College of Pharmacy, Karad in 2001. Dissertation topic: “Therapeutic Drug Monitoring of Phenytoin”. Worked as Principal for One year at College of Pharmacy, Medha. Presently working as Lecturer at Sinhgad Institute of Pharmaceutical Sciences, Lonavala.

Various papers are presented in Indian Pharmaceutical Congress (IPC) and International Convention of Association of Pharmaceutical Teachers of India (APTI).

Life Member of APTI. Area of interest: Novel Drug Delivery System and Clinical Pharmacology.

* Corresponding author, Sinhgad Technical Education Society’s Sinhgad Institute of Pharmaceutical Sciences,

309/310, Off Mumbai-Pune Expressway, Kusgaon (Bk), Lonavala, Tal-Maval, Dist-Pune , Pin: 410 401

Phone 02114-280076, 280205 Mobile: 9822628406 e-mail: sbb2000@rediffmail.com

Prof. Kane R. N.

Completed M. Pharm in Medicinal & Pharmaceutical Chemistry from Dept of Pharmacy, S.G.S.I.T.S. Indore, Rajiv Gandhi Technological University, Bhopal. Dissertation topic “3-D QSAR Analysis of 2,3 diaryl cyclopentenones as selective Cyclooxygenase 2 inhibitor”.11 years experience as lecturer and 3 years experience as Principal. Presently working as Principal at Sinhgad Institute of Pharmaceutical Sciences, Lonavala.

Various papers are presented in Indian Pharmaceutical Congress (IPC) and International Convention of Association of Pharmaceutical Teachers of India (APTI). Life Member of APTI. Area of interest: Novel Drug Delivery System.

Dr. Bhise S. B.

Completed M. Pharm from Haffkin Institute of Training, Research & Testing, Mumbai in 1977.

Completed Ph.D. in Pharmacology from Birla Institute of Technology, Pilani in 1983. Experience: CRA (2 Yrs), Lecturer (6 Yrs), Assistant professor (3 Yrs), Principal (17 Yrs)

Expert Member of AICTE, PCI, MS, Editorial Board of IJPE.

Research Paper Published: 44 (Research 29 + Review 5 + Others 10)

Books Published: 3

Life Member of APTI, ISTE, IPS, IIPA, IPA, IHPA, NCQM, ITDMS, ACS, IATDMCT.

Area of interest: Bioavailability & Bioequivalance and Clinical Pharmacology.