koleksi stem-cells-1

EDITED BY ;PROF.DR.PRATIWI TS,MS

04/12/23STEM CELLS -PROF PRATIWI 20121

KOLEKSI DAN APLIKASI STEM SEL

04/12/232 STEM CELLS -PROF PRATIWI 2012

Stem Cell – Definition


A cell that has the ability to continuously divide and differentiate (develop) into various other kind(s) of cells/tissues

Introduction


Can we vs. should weDramatic advances of modern molecular

geneticsShould we ask the morality questions

before attempting the “can we” questions?

Stem Cell/Cloning Topics


What are stem cells?History of stem cell/cloning researchPossible uses of the technologyCurrent status/knowledgeQuestions and known problemsLegal considerationsPoliticsMoral considerations

Kinds of Stem Cells

Stem cell type Description Examples

Totipotent Each cell can develop into a new individual

Cells from early (1-3 days) embryos

Pluripotent Cells can form any (over 200) cell types

Some cells of blastocyst (5 to 14 days)

MultipotentCells differentiated, but can form a number of other tissues

Fetal tissue, cord blood, and adult stem cells


Totipotent

Pluripotent

Multipotent

Stem Cell/Cloning Topics


What are stem cells?History of stem cell/cloning researchPossible uses of the technologyCurrent status/knowledgeQuestions and known problemsLegal considerationsPoliticsMoral considerations

Kinds of Stem Cells

Stem cell type Description Examples

Totipotent Each cell can develop into a new individual

Cells from early (1-3 days) embryos

Pluripotent Cells can form any (over 200) cell types

Some cells of blastocyst (5 to 14 days)

MultipotentCells differentiated, but can form a number of other tissues

Fetal tissue, cord blood, and adult stem cells


Totipotent

Pluripotent

Multipotent

Stages of Embryogenesis


Day 1Fertilized egg

Day 1Fertilized egg

Day 22-cell embryo

Day 22-cell embryo

Day 3-4Multi-cell embryo

Day 3-4Multi-cell embryo

Day 5-6BlastocystDay 5-6

BlastocystDay 11-14Tissue Differentiation

Day 11-14Tissue Differentiation

Derivation and Use of Embryonic Stem Cell Lines


Isolate inner cell mass(destroys embryo)

Isolate inner cell mass(destroys embryo)

Heart muscleKidney

Liver

“Special sauce”(largely unknown)

Day 5-6BlastocystDay 5-6

Blastocyst

Inner cells(forms fetus)Inner cells

(forms fetus)

Outer cells(forms placenta)

Outer cells(forms placenta)

Heartrepaired

Culture cellsCulture cells

Bone Marrow Stem Cells


Possible Uses of Stem Cell Technology


Replaceable tissues/organsRepair of defective cell typesDelivery of genetic therapiesDelivery chemotherapeutic agents

Early Successes – Adult Stem Cells


Human mesenchymal stem cells turned on genes found in bone, cartilage, adipose, muscle, hematopoiesis-supporting stromal, endothelial, and neuronal cells.

Multipotent adult progenitor cells have been shown to differentiate into functional, hepatocyte-like cells.

Early Successes – Adult Stem Cells


Human neural stem cells can migrate extensively in the brain after injection.

Adult stem cells have been isolated from amniotic fluid, peripheral blood, umbilical cord blood, umbilical cord, brain tissue, muscle, liver, pancreas, cornea, salivary gland, skin, tendon, heart, cartilage, thymus, dental pulp, and adipose tissue.

Early Successes – Human Cloning


2001 – First cloned human embryos (only to six cell stage) created by Advanced Cell Technology (USA)

2004* – Claim of first human cloned blastocyst created and a cell line established (Korea) – later proved to be fraudulent

*Hwang, W.S., et al. 2004. Evidence of a Pluripotent Human Embryonic Stem Cell Line Derived from a Cloned Blastocyst. Science 303: 1669-1674.

Cloned Embryonic Stem Cells –

Advantages/Problems


AdvantagesNo rejection“Prefect match”

ProblemsOnly 10% of cloned oocytes became embryos

0% (0 out of 2061) survived to become a cell line

Genetic donor was same as egg donor (i.e., won’t work for males!)

Cost is high (health insurance probably won't pay)

Challenges to Stem Cell/Cloning Research


Stem cells need to be differentiated to the appropriate cell type(s) before they can be used clinically.

Recently, abnormalities in chromosome number and structure were found in three human ESC lines.



Stem cell development or proliferation must be controlled once placed into patients.

Possibility of rejection of stem cell transplants as foreign tissues is very high.



Contamination by viruses, bacteria, fungi, and Mycoplasma possible.

The use of mouse “feeder” cells to grow ESC could result in problems due to xenotransplantation (complicating FDA requirements for clinical use).

At Conception, It Is Only a Single Cell


Claim:Fertilized eggs are single cells, like blood

cells or other parts of the bodyRebuttal:This single cell is unique from both the

father’s and mother’s cells and is the beginning of every new human being

Only a Small Percentage of Embryos Implant


Claim:Embryos are only potential life. Most do not result

in birthsRebuttal:25-33% of women become pregnant in the first

month33% of implanted embryos die before birthThere are countries in which over 25% of children

die before age 5. Should we allow killing of children?

Unexpected Phenotypes

Phenotype more severe than expected:- Early lethal- Lack of inductive signalsPhenotype less severe than expected:- Incomplete gene disruption- Genetic redundancy- Functional redundancy (compensation)


Applied research - DNA

Applied research arising out of the discovery of DNA includes disease diagnosis, drug development, gene therapy and, more recently, genetically-modified organisms


Transgenic Animals and Products

Mice- transgenetic mice have been used in several ways.

One of the best known is to produce human antibodies.

Cattle- are used to control disease such as mastitis in dairy cows.


Methods of creating transgenetic animals

Step One- collect embryosWith proper stimulation far more embryos can be obtained than would be the natural result of the reproductive process.


Methods of creating transgenetic animals

Step Two- Inject embyros.A pro nucleus is the haploid nucleus of the sperm or ovum that have united in fertilization to form a zygote.


Embryo Transfer

Embryo transfer is the harvesting of fertilized ova from a donor and implanting them into a recipient.

The harvested embyros are transferred to a recipient.


Clone Birth Defects

• Cloned offspring often suffer from large offspring syndrome, where the clone and the placenta that nourished it are unusually large.

• Cloned offspring often have serious inexplicable respiratory or circulatory problems, which causes them to die soon after birth.

• Clones tend to have weakened immune systems and sometimes suffer from total immune system failure.

• Very few clones actually survive to adulthood.



Clones appear to age faster than normal.

Clones experience problems associated with old age, such as

arthritis, while they are still young. This may be due to the fact that clones

have shorter telomeres

Transgenic Animals:

Animal biotechnology is the field to engineer transgenic animals, i.e., animals that carry genes from other species.

The technology has already produced transgenic animals such as mice, rats, rabbits, pigs, sheep, and cows.


Transgenic Animals

Definition: An organism (typically a mouse) that is engineered to carry a foreign gene, or transgene of choicem as part of its own genetic material.


Transgenic Animals

Purpose: These animals are very useful for delineating the function of newly discovered genes as well as for producing useful proteins in large animals.


Transgenic Animals

In some of the eggs, the genetic material integrates at a random site on a chromosome and so becomes part of the mouse cell's genetic material the animal resulting from that egg will therefore carry that gene and so is referred to as a "transgenic animal".


What is a transgenic animal?

A transgenic animal is one whose genome has been changed to carry genes from other species.

For example, an embryo can have an extra, functioning gene from another source artificially introduced into it, or a gene introduced which can knock out the functioning of another particular gene in the embryo.


Transgenic Animals

Animals that have their DNA manipulated in this way are known as transgenic animals.

Transgenic animals are useful as disease models and producers of substances for human welfare.


Why are these animals being produced?

Some transgenic animals are produced for specific economic traits.

E.g., transgenic cattle were created to produce milk containing particular human proteins, which may help in the treatment of human emphysema.


How are transgenic animals produced?

DNA microinjectionIntroducing the transgene DNA directly into the zygote at an early stage of development.

No vector required.

Retrovirus-mediated gene transfer: Infecting mouse embryo with a retrovirus

which carry the new gene. Using virus as a vector .


Embryonic stem cell-mediated gene transfer The blastocyst (inner layer of a fertilized

egg) is harvested and mixed with recombinant DNA and inserted back in the blastocyst.

Sperm-mediated transfer

Use of “Linker protein" to attach DNA to sperm which transfer the new DNA during fertilization.

Gene gun


Embryonic stem cell-mediated gene transfer

This method involves: Isolation of totipotent stem cells (stem cells

that can develop into any type of specialized cell) from embryos.

The desired gene is inserted into these cells. Cells containing the desired DNA are

incorporated into the host's embryo.


First Breeding Pair:Fertile male + superovulated female

Fertile male Superovulated female = immature female

induced to superovulate Pregnant mare’s serum (=FSH) on day 1 Human Chorionic Gonadotropin (=LH) on day 3

Mated on day 3Fertilized oocytes microinjected on day 4 with

foreign DNA construct.Microinjected oocytes are transferred to the

oviducts of surrogate mothers at end of day 4.

Procedure for Producing Transgenic Mice


Second breeding pair:Sterile male + surrogate mother

Sterile male produced through vasectomySurrogate mother must mate to be

suitable recipient of injected eggsMated on day 3Microinjected oocytes from first breeding

pair are transferred to oviducts on day 4Embryos implant in uterine wall and are

born 19 days later.Southern blotting techniques confirm

presence and copy number of transgenes.



Third breeding pair:Foster parents

Fertile male + female mated to give birth on same day surrogate mother

Serves as foster parent if caesarian section is required for surrogate mother



Totipotent and pluripotent cells

Totipotent =meaning that its potential is total.

pluripotent = they can give rise to many types of cells but not all types of cells (no fetus developed).

isolated directly from the inner cell mass of embryos at the blastocyst stage.

(IVF-IT surplus embryos in case of humans)


More about stem cells

Embryonic stem cells Adult stem cells

Truly pluripotential More restricted pattern of differentiation

medical gain without ethical pain

several countries have sanctioned deriving

human ES-cell lines from ‘surplus’ embryos

created through in vitro fertilization

although several humanES-cell lines have been made,

they will not be immunologically compatible with most patients

who require cell transplants.04/12/2349 STEM CELLS -PROF PRATIWI 2012

Transgenic miceThe growth hormone gene has been engineered to be expressed

at high levels in animals.

The result: BIG ANIMALS

metallothionein promoter regulated as heavy metals

Mice fed heavy metals are 2-3 times larger


Studies Utilizing Transgenic Mice

“Pharm” animals (transgenic livestock)

Bioreactors whose cells have been engineered to synthesize marketable proteins

DNA constructs contain desired gene and appropriate regulatory sequences (tissue-specific promoters)

More economical than producing desired proteins in cell culture


Antifreeze gene promoter with GH transgene in atlantic salmon

GH gene comes from larger salmon


Wild and domestic trout respond differently to overproduction of growth hormone.

So in some cases, GH not effective.


Improving Agricultural Products with Transgenics

Transgenic technology holds great potential in agriculture, medicine, and industry

The benefits of these animals to human welfare can be grouped into areas:

Agriculture Medicine Industry


1. Agricultural Applications

A) Breeding Traditional cross breeding have been

used for ages to create chickens, cows, pigs etc.

Farmers have always used selective breeding to produce animals that exhibit desired traits (e.g., increased milk production, high growth rate).

Traditional breeding is a time-consuming, difficult task.


Researchers have now used gene transfer to improve the productivity of livestock.

Now it is possible to develop traits in animals in a shorter time and with more precision.

It also offers farmers an easy way to increase yields.


Scientists can improve the size of livestock genetically.

Transgenic cows exist that produce more milk or milk with less lactose or cholesterol.

Transgenic cows have been used to produce milk which are richer in proteins and lower in fat.


B) Quality Herman, a transgenic bull carries a

human gene for Lactoferrin (gene responsible for higher iron content)

Pigs and cattle that have more meat on them.

Sheep that grow more wool. Eggs can be made healthier with high

quality protein.


C) Disease resistance Disease-resistant livestock is not a

reality just yet. But there has been improvement in

disease reduction in animals. The Foot- and- Mouth disease in

England in 2000 led to destruction of herds of cattle, sheep and goat.


Scientists are attempting to produce disease-resistant animals, such as influenza-resistant pigs, but a very limited number of genes are currently known to be responsible for resistance to diseases in farm animals.

Transgenic disease protection promises a long term cost effective method of battling animal diseases.


2. Medical Applications

A) Xenotransplantation

Transplant organs may soon come from transgenic animals.


B) Nutritional supplements and pharmaceuticals

Products such as insulin, growth hormone, and blood anti-clotting factors may soon be or have already been obtained from the milk of transgenic cows, sheep, or goats.

The first transgenic cow (Rosie ) produced human protein-enriched milk at 2.4 grams per liter.


This transgenic milk is a more nutritionally balanced product than natural milk and could be given to babies or the elderly with special nutritional or digestive needs.

A transgenic cow exists that produces a substance to help human red cells grow.


C) Human gene therapy

Human gene therapy involves adding a normal copy of a gene (transgene) to the genome of a person carrying defective copies of the gene.

Finland produced a calf with a gene that makes the substance that promotes the growth of red cells in humans.


3. Industrial Applications :

By extracting polymer strands from the milk and weaving them into thread, the scientists can create a light, tough, flexible material that could be used in such applications as military uniforms, medical microsutures, and tennis racket strings.

Biosteel is an extraordinary new product that may be soon used in bullet proof vests and in suture silk for stitching wounds.

Animals have been used as “Bioreactors” to produce proteins. Genes for desired proteins are introduced via transgenics to the target cells .


:

The target cells are cloned and several such cells are raised into adults.

These adults may produce milk or eggs (due to the presence of introduced gene rich in desired protein).

Toxicity-sensitive transgenic animals have been produced for chemical safety testing.

Microorganisms have been engineered to produce a wide variety of proteins, which in turn can produce enzymes that can speed up industrial chemical reactions.


Transgenic animals have been used to produce pharmaceutical protein: example a human gene called AT III has been transferred to goats.

Goats milk contain this protein that prevents blood clotting (goats multiply faster than cows)

“Hen bioreactor” eggs are used to enrich protein by recombinant DNA technology.


Transgenic Goats That Produce Valuable Proteins in Their Milk – “Biorectors”


A Summary of Animal Cloning

Although there has been limited success in cloning some animals, it's still seen as a viable technology.

Ever since the announcement of the birth of Dolly, additional sheep, cows, goats, pigs, and mice have been cloned.


:

There are still obvious problems as evidenced from the numerous deaths of cloned animals that occur just before or after birth.

Cloning is a big first step. Genetic manipulation of cloned animals is the future direction of the cloning frontier.


koleksi stem-cells-1

Technology

stem cells prof pratiwi

blood cells

cells of blastocyst

human mesenchymal stem

human neural stem cells

single cells

bone marrow stem cells

neuronal cells