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The ethical and moral debate over the use of stem cells has taken center stage over the past decade. Stem cells are of great medical interest, since they have the potential to develop into almost any type of cell in the body. Regenerative medicine focuses on the potential uses of stem cells in medicine and how they can provide effective treatment for a range of diseases.

Stem cells have the capacity to divide indefinitely to replenish other cells in the body. When a stem cell divides, each daughter cell can remain a stem cell or become a more specialized cell, such as a red blood cell, a muscle cell or a nerve cell. An increasing body of evidence also suggests that molecular pathways and properties associated with normal stem cells is relevant to cancer development [1].

There are two types of mammalian stem cells: embryonic stem cells, which are found in blastocysts (the mammalian embryo at the stage at which it is implanted into the wall of the uterus), and adult stem cells, which are found in adult tissues.

Stem cells are undifferentiated, meaning that they aren’t committed to becoming a specific kind of cell in the body. They are characterized by the ability to self-renew (remaining undifferentiated) and the capacity to differentiate into specialized cell types (termed potency). Potency specifies a stem cell’s differentiation potential.

There are four classes of stem cell potency:

• Totipotent: A totipotent stem cell is produced from the fusion of an egg and sperm cell and has the ability to become any kind of cell in the body. For a brief period, each cell division creates identical totipotent cells — any one of these cells could develop into a fetus if placed in a woman’s uterus. By the fourth day, the totipotent cells begin to specialize, forming a blastocyte, the type of cell that forms the outer layer of a blastocyst. These cells will go on to form the placenta and other supporting tissues of the uterus necessary for fetus development.

• Pluripotent: The inner cluster of cells in a blastocyst, called inner mass cells, are pluripotent. Pluripotent stem cells have the ability of to become any kind of cell in the body other than cells of the placenta or other supporting tissues of the uterus. Because of this, pluripotent cells cannot form a fetus if placed in a woman’s uterus. Embryonic stem cells are generally considered pluripotent.

• Multipotent: As pluripotent cells continue to specialize, they become cells that only lead to the development of specific tissues. Multipotent stem cells have the ability to form several cell types of a closely related family of cells and are generally referred to by their tissue origin, e.g. cardiac stem cells, neural stem cells, bone marrow stem cells. Multipotent cells function as a repair system for damaged tissue. Adult stem cells are multipotent stem cells.

• Unipotent: Unipotent stem cells have the ability to produce only one specialized cell type. Unipotent cells maintain the property of self-renewal, thus distinguishing them from non-stem cells.

Pluripotent stem cells can be derived from a number of sources, all of which are associated with moral issues:

1. Surplus embryos that are the by-product of in vitro fertilization
2. Embryos created in the lab from donated sperm and eggs
3. Embryos created through a cloning technique called somatic cell nuclear transfer
4. Embryos from aborted fetuses

Pluripotent stem cells can also be obtained from umbilical cord blood, placenta and amniotic fluid.

Although pluripotent stem cells have greater potential, multipotent adult stem cells are currently being used therapeutically. Adult stem cells can be found in many organs and tissues, including bone marrow, peripheral blood, brain, muscle, liver, skin and heart. Nevertheless, a number of limitations exist with adult stem cells [2]. Adult stem cells are found in small quantities in adult tissues and umbilical cord blood, raising doubt that they could be grown in clinically significant quantities. Additionally, adult stem cells have not been found for all tissues of the body, necessitating the use of pluripotent stem cells for the generation of certain tissue types. Since adult stem cells are multipotent, they cannot be induced to develop into any cell type, i.e. manipulated to differentiate into a specialized cell type different than their final tissue type.

For more information on stem cell research and current federal policy on embryonic stem cell research, see Research!America.

References

1. Lobo et al. The biology of cancer stem cells. Annu Rev Cell Dev Biol. 2007;23:675-99.
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2. Vats et al. Stem cells. Lancet. 2005 Aug 13-19;366(9485):592-602.
   View abstract

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What does the diagnosis of hereditary diseases, the detection and diagnosis of infectious diseases, personalized DNA sequencing, DNA cloning, genetic functional analysis, genetic fingerprinting and DNA-based phylogeny have in common?

The all employ a widely used molecular technique called polymerase chain reaction or PCR.

The idea was conceived by Kary Mullis in the early 1980s and was first described, albeit briefly, in an article investigating the mutation that causes sickle cell anemia [1]. The details of the method and its uses were discussed in greater detail over the next two years [2-3]. PCR revolutionized molecular genetics by allowing rapid duplication and analysis of DNA.

The PCR method

PCR is used to amplify a specific region of DNA in order to produce a large number of nearly identical copies. The method uses a heatstable DNA replication enzyme called a DNA polymerase, the four deoxynucleotide building blocks of DNA and two small single-stranded DNA segments called primers, which flank the “target” region of DNA to be amplified and are complementary to each strand (meaning the matching strand to which its bases pair).

There are 3 basic steps in PCR that are carried out at different temperatures to create conditions optimal for:

1. DNA denaturation (meaning to separate the double-stranded DNA into single strands).
2. Primer binding or hybridization to each of the single strands of DNA at either the beginning or the end of the target sequence, depending upon the single-strand of DNA. Hybridization combines complementary, single-stranded DNA into a single molecule. This process is called annealing.
3. DNA polymerase elongation. The enzyme attaches to the primer-single-stranded DNA duplex and synthesizes the complementary strand of DNA, using the existing single-strand as a template.

Newly synthesized DNA strands can serve as additional template for complementary strand synthesis. PCR rapidly amplifies DNA; because both strands are copied, there is an exponential increase in the number of copies. Assuming there is only a single copy of the target gene before cycling starts:

After 35 cycles of PCR, there will be over 68 billion copies! In reality, PCR starts with many copies of the target gene, so the end result is typically higher. Each cycle only takes a few minutes. Factoring in the time to change temperatures, the entire process can be done in several hours.

More recently, a new method of PCR quantification called real-time PCR or quantitative real-time PCR (qRT-PCR) was developed [4]. qRT-PCR enables the detection and quantification of a specific DNA sequence using a fluorescent reporter (either a dye or a modified DNA oligonucleotide probe), which increases in direct proportion to the amount of DNA amplified in a reaction.

It’s easier to understand how PCR works with pictures. Visit DNA Interactive to view an animation of PCR. DNA Interactive is an award-winning educational web site created in 2003 to celebrate the 50th anniversary of the discovery of the DNA double helix structure.

The advent of PCR and recombinant DNA technologies have enabled numerous applications in basic and clinical research. PCR is often regarded as one of the most important scientific advances in molecular biology. Indeed, Kary Mullis holds the only Nobel Prize ever awarded to a scientist in the biotechnology industry. He received the Nobel Prize for Chemistry in 1993.

References

1. Saiki et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350-4.
   View abstract
2. Mullis et al. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol. 1986;51 Pt 1:263-73.
   View abstract
3. Mullis and Faloona. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335-50.
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4. Higuchi et al. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology (N Y). 1993 Sep;11(9):1026-30.
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Mitosis is the biological process involving chromosomal duplication and nuclear division. Mitosis is usually followed by cytokinesis, whereby the watery environment inside a cell, known as the cytoplasm, and cell membrane divide. Two identical cells are generated, each having the same number of chromosomes as the parental cell. Somatic cells (meaning any cell that is not a germline cell) undergo mitosis while germ cells (cells destined to become sperm or eggs) divide by a related process called meiosis.

The biological process of Meiosis involves DNA synthesis followed by two rounds of cell division and results in four daughter cells with half the number of chromosomes as the starting cell. Meiosis is essential for sexual reproduction. Meiotic recombination, also known as crossing over, is the process in which two chromosomes exchange segments of DNA and is responsible for genetic variation.

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