Life of a Cell• Cells will replicate normally for an allotted period of time • Telomeres are the end structures of eukaryotic chromosomes that guide the number of times a cell can replicate• In humans, consists of specific repeating sequences (TTAGGG)• 8,000 - 12,000 base pairs• Chromosome “caps”• Most human body cells can divide maximum of about 50 - 90 times

Senescence• Telomeres guide cellular senescence (aging process) • Each time a cell divides, telomeres shorten slightly• DNA polymerase is prevented from completely copying the ends of both strands• Multiple copies of repeating sequences allows cells to lose DNA without affecting gene sequences

Cancer

• Cancer cells are immortal (divide indefinitely)• Telomerase is an enzyme that repairs telomere length at the ends of chromosomes by adding DNA nucleotides to cap telomere after each round of cell division• Not active in normal cells, but active in over 90% of cancers

Fountain of Youth?• Telomere shortening is involved in the aging process• Wrinkles, arthritis, gray hair, & other signs of aging• Telomerase is NOT a “cure” for aging• Researchers are looking at the inhibition of telomerase as an anticancer vaccine target

Telomerase genes• Human cells with active telomerase genes would display immortality• Valuable for treating individuals with age-related disorders (arthritis, neurodegenerative disorders)• Perhaps introduce telomerase genes into aging cells and return them (extending life span)• Another cell type containing telomerase is a stem cell

Stem Cell Technologies• CDC indicates that ~3,000 Americans die every day from diseases that may one day be treated by stem cells• Most stem cells are taken from embryos• We must look at development of an embryo to understand stem cells• Fertilization of an egg cell with sperm creates a zygote • In vitro fertilization (IVF) may also be used to create a zygote

In vitro fertilization (IVF)• IVF is an assisted reproduction technology in which sperm & egg cells are removed from patients and cultured in a dish to achieve fertilization• After several days of division, the embryo is surgically implanted into the uterus• Remaining embryos are frozen for future use (a potential source of embryonic stem cells)

Embryonic Development• The zygote rapidly divides and after 3-5 days forms a morula (compact ball of about 12 cells)• About 5-7 days after fertilization, dividing cells create a blastocyst (an embryo consisting of a small hollow cluster of about 100 cells)• The blastocyst is approximately 1/7 mm in diameter

Blastocyst• The outer row of single cells of the blastocyst is called the trophoblast, which develops to form part of the placenta that nourishes the embryo• The small cluster of about 30 cells tucked inside the blastocyst form the inner cell mass (ICM)• The cells of the ICM are the source of human embryonic stem cells (ES cells)

ICM

T

Stem Cells• Cells of the ICM have the ability to undergo differentiation (cellular specialization)• Stem cells can differentiate to form all of the more than 200 cell types in the human body• Stem cells are defined based on 2 properties:

1. Infinite self-renewal (develop other stem cells)

2. Unlimited potency (can develop into a variety of cell types)

Cellular Potency• Totipotent cells can become any type of cell; total potential (ex. embryo, morula)• Pluripotent cells can develop into any bodily tissue but not extraembrionic tissue (ex. cells of ICM)• Multipotent cells can develop into cells with specific functions limited in number (ex. bone marrow, umbilical cord)

Work on ES cells• 1st successful isolation & culturing of ES cells reported in 1998 by James Thomson (U. of Wisconsin)• He had isolated stem cells of rhesus monkey 2 years earlier• Many scientists credit their work to experiments done on mice in the 1980s (another example of model organisms advancing science)

ES cell Characteristics

• Human ES cells avoid senescence because of increased levels of telomerase• Grow rapidly• Can be frozen for long periods of time• Growth factors & other signals coax them to differentiate into various cell types

ES cell Research• Human ES cells for research are derived from blastocysts of embryos that are no longer needed by couples for IVF (or donations to create embryos)• Another source is by somatic cell nuclear transfer (SCNT - therapeutic cloning)• Research is controversial because of the source of ES cells • Some opponents of ES cell research claim adult derived stem cells are more acceptable

Adult-derived Stem Cells (ASCs)

• ASCs can be cultured & differentiated to produce other cell types (ex. muscle to blood)•Harvested from fine-needle biopsy, hence no need for human embryos• However - Not discovered in all tissues (brain, intestines, skin, muscle, & blood so far) •Appear in small #s•May not be as pluripotent

Parthenogenesis• Parthenogenesis is the process of creating an embryo without fertilization• Human egg cells treated to retain 46 chromosomes to induce embryonic development from egg• Occurs naturally in salamanders and certain other animals• Do not appear to be able to develop to form full-term babies• Stem cells from parthenogenic primate eggs already isolated

Stem Cell Applications• Grow healthy tissues• Genetically manipulate stem cells for gene delivery• However, potential and promise are key words since these applications are yet unproven• Applications in leukemia, blood deficiencies, & trauma amongst others

Leukemia• Leukemia patients require chemotherapy and radiation• White blood cells (WBCs) divide abnormally• May require transfusions• Using stem cells to make WBCs becoming effective to treat

Blood Deficiencies• Stem cells from umbilical cord used to provide red blood cells (RBCs) for sickle cell patients and others with blood deficiencies• Becoming popular• Parents can opt to pay to have cord blood stem cells frozen indefinitely should child need them in the future

Trauma• Stem cells may be used to replace dead/dying cells following trauma (ex. heart attack)• Death of cardiac muscle cells weakens heart• Adult cardiac cells do not repair themselves well• In mice, scientists injected ASCs from mouse bone marrow into damaged area of heart

• Developed into cardiac muscle cells• Formed electrical connections with healthy cells• Improved heart function by over 35%

Potential• ASCs isolated from brain & even nose can be used to make neurons in culture• ES cells can be differentiated to form neurons that can be injected to improve neural function in animals with spinal cord injuries• Human stem cell transplants can enable mice with paralyzed hind limbs to walk

Paralysis in Rats

Quadriplegic Walks

Mouse Stem Cell Experiment

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Stem Cells

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Quadriplegic Walks

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Unresolved Questions

• Is there an “ultimate” adult stem cell?• Why do stem cells self-renew and remain undifferentiated?• What factors trigger division?• What growth factors influence differentiation?• What factors affect integration of new tissues and cells into existing organs?

Cloning• Cloning refers to making a copy of something - a gene, a cell, or an entire organism• Employs rDNA technology• Two approaches to cloning:

• reproductive cloning, which intends to create a baby (ex. Dolly)• therapeutic cloning, which provides stem cells that are a genetic match to a patient (aka somatic cell nuclear transfer - SCNT)

Cloning Process

• Nucleus of a somatic cell isolated• Injected into an enucleated egg (programmed for development)•Egg stimulated to divide in culture to create an embryo (implanted in uterus in reproductive cloning)• Embryo grown until it reaches blastocyst stage (to harvest stem cells)• Stem cells isolated, grown in culture, then introduced into patient

Cloning cont’d• Therapeutic cloning may be able to treat disease without a fear of immune rejection• Stem cells from patient may be able to create stem cell lines from humans with genetic disease (to study & learn more)• However, many scientists think that reproductive cloning of humans is unethical, immoral, & scientifically unsafe

Human cloning• Many private groups have announced plans about intentions to clone human embryos• US legislation prevents federal funding of cloning research• Advanced Cell Technology (ACT) is a private Massachusetts biotech company that announced in Nov. 2001 that they cloned 1st human embryos using somatic cell nuclear transfer• Many scientists have dismissed ACT claims

Human Genome Project & Disease Genes

• Scientists have been developing complex “maps” showing the locations of normal & diseased genes on all human chromosomes• Cloning and sequencing technologies have enabled such a project to exist• The depth of this knowledge will take us to many unknown heights, but will still leave many valleys untouched