stem cells lecture dr. ziad jaradat. dr. ziad w jaradat © stem cells the history of research on...

Stem cells lectureStem cells lecture

Dr. Ziad JaradatDr. Ziad Jaradat

Dr. Ziad W Jaradat ©Dr. Ziad W Jaradat ©

STEM CELLSSTEM CELLS

The history of research on adult stem cells The history of research on adult stem cells began about 40 years ago. In the 1960s, began about 40 years ago. In the 1960s, researchers discovered that the bone researchers discovered that the bone marrow contains at least two kinds of stem marrow contains at least two kinds of stem cells. One population, called cells. One population, called hematopoietic stem cellshematopoietic stem cells, forms all the , forms all the types of blood cells in the body. A second types of blood cells in the body. A second population, called population, called bone marrow stromal bone marrow stromal cellscells, was discovered a few years later. , was discovered a few years later.

Stromal cellsStromal cells are a mixed cell population are a mixed cell population that generates bone, cartilage, fat, and that generates bone, cartilage, fat, and fibrous connective tissue. fibrous connective tissue.


Where are adult stem cells found and Where are adult stem cells found and what do they normally do?what do they normally do?

An adult stem cell is an An adult stem cell is an undifferentiatedundifferentiated cell found among cell found among differentiated cells in a tissue or organ, differentiated cells in a tissue or organ, can renew itself, and can differentiate can renew itself, and can differentiate to yield the major specialized cell types to yield the major specialized cell types of the tissue or organ. of the tissue or organ.


Where adult stem cells are Where adult stem cells are found?found?

Adult stem cells have been identified in many organs Adult stem cells have been identified in many organs and tissues. However, there are a very small and tissues. However, there are a very small number of stem cells in each tissue. number of stem cells in each tissue.

Stem cells are thought to reside in a specific area of Stem cells are thought to reside in a specific area of each tissue where they may remain quiescent (non-each tissue where they may remain quiescent (non-dividing) for many years until they are activated by dividing) for many years until they are activated by disease or tissue injury. disease or tissue injury.

The adult tissues reported to contain stem cells The adult tissues reported to contain stem cells include include brainbrain, , bone marrowbone marrow, , peripheral bloodperipheral blood, , blood blood vessels, skeletal muscle, skinvessels, skeletal muscle, skin and and liverliver..


Role of adult stem cells Role of adult stem cells The primary role of The primary role of adult stem cellsadult stem cells in a in a

living organism are to living organism are to maintain and repair the maintain and repair the tissuetissue in which they are found. in which they are found.

Some scientists now use the term Some scientists now use the term somatic somatic stem cellstem cell instead of adult stem cell. Unlike instead of adult stem cell. Unlike embryonic stem cellsembryonic stem cells, which are defined by , which are defined by their origin (the their origin (the inner cell massinner cell mass of the of the blastocystblastocyst), the origin of adult stem cells in ), the origin of adult stem cells in mature tissues is mature tissues is unknownunknown..


What are the tests used for identifying adult stem What are the tests used for identifying adult stem cells?cells?

One or more of the following methods are normally One or more of the following methods are normally used for the identification of adult stem cells:used for the identification of adult stem cells:

• Labeling the cellsLabeling the cells in a living tissue with molecular markers and in a living tissue with molecular markers and

then determining the specialized cell types they generate. then determining the specialized cell types they generate.

• Removing the cells from a living animalRemoving the cells from a living animal, labeling them in cell , labeling them in cell culture, and transplanting them back into another animal to culture, and transplanting them back into another animal to determine whether the cells repopulate their tissue of origindetermine whether the cells repopulate their tissue of origin

• Isolating the cellsIsolating the cells, growing them in cell culture, and , growing them in cell culture, and

manipulating them, often by adding growth factors or manipulating them, often by adding growth factors or introducing new introducing new genesgenes, to determine what differentiated cells , to determine what differentiated cells types they can become.types they can become.


A single adult stem cell should be able to A single adult stem cell should be able to generate a line of genetically identical generate a line of genetically identical cells—known as a cells—known as a cloneclone—which then —which then gives rise to all the appropriate gives rise to all the appropriate differentiated cell types of the tissue. differentiated cell types of the tissue.

Adult stem cells may exhibit two types of Adult stem cells may exhibit two types of differentiation pathways; differentiation pathways; • Normal differentiationNormal differentiation; to form specialized ; to form specialized

cell types of the tissue in which they reside.cell types of the tissue in which they reside.• TransdifferentiationTransdifferentiation or or plasticityplasticity; ; cells may cells may

also exhibit the ability to form specialized cell also exhibit the ability to form specialized cell types of tissues types of tissues other thanother than the place where the place where they reside.they reside.


Normal DifferentiationNormal DifferentiationThe following are examples of differentiation The following are examples of differentiation

pathways of adult stem cells pathways of adult stem cells ((FigureFigure 1 1).).

• Hematopoietic stemHematopoietic stem cells give rise to all types of cells give rise to all types of blood cellsblood cells: red blood cells, B lymphocytes, T : red blood cells, B lymphocytes, T lymphocytes, natural killer cells, neutrophils, lymphocytes, natural killer cells, neutrophils, basophils, eosinophils, monocytes, macrophages, basophils, eosinophils, monocytes, macrophages, and platelets. and platelets.

• Bone marrow stromalBone marrow stromal cells ( cells (mesenchymal stem mesenchymal stem cellscells) give rise to a variety of cell types: bone ) give rise to a variety of cell types: bone cells (osteocytes), cartilage cells (chondrocytes), cells (osteocytes), cartilage cells (chondrocytes), fat cells (adipocytes), and other kinds of fat cells (adipocytes), and other kinds of connective tissue cells such as those in tendons. connective tissue cells such as those in tendons.


• Neural stemNeural stem cells in the brain give rise to its three cells in the brain give rise to its three major cell types: major cell types: nerve cells nerve cells (neurons) and two (neurons) and two categories of non-neuronal cells- categories of non-neuronal cells- astrocytesastrocytes and and oligodendrocytesoligodendrocytes..

• Epithelial stemEpithelial stem cells in the lining of the digestive cells in the lining of the digestive tract occur in deep crypts and give rise to several tract occur in deep crypts and give rise to several cell types: absorptive cells, goblet cells, Paneth cell types: absorptive cells, goblet cells, Paneth cells, and entero-endocrine cells. cells, and entero-endocrine cells.

• Skin stemSkin stem cells occur in the basal layer of the cells occur in the basal layer of the epidermis and at the base of hair follicles. The epidermis and at the base of hair follicles. The epidermal stem cells give rise to keratinocytes, epidermal stem cells give rise to keratinocytes, which migrate to the surface of the skin and form which migrate to the surface of the skin and form a protective layer. The follicular stem cells can a protective layer. The follicular stem cells can give rise to both the hair follicle and to the give rise to both the hair follicle and to the epidermis.epidermis.


Trans-differentiation or plasticity of adult Trans-differentiation or plasticity of adult stem cellsstem cells

Certain adult stem cell types are Certain adult stem cell types are pluripotentpluripotent. This ability to . This ability to differentiate into differentiate into multiple cell typesmultiple cell types is called is called plasticity or plasticity or transdifferentiationtransdifferentiation. .

The following list offers examples of adult stem cell The following list offers examples of adult stem cell plasticity that have been reported during the past plasticity that have been reported during the past few years (few years (Figure 2Figure 2).).

Hematopoietic stem cells may differentiate into: Hematopoietic stem cells may differentiate into: • three major types of brain cells (neurons, oligodendrocytes, and three major types of brain cells (neurons, oligodendrocytes, and

astrocytes) astrocytes) • skeletal muscle cells skeletal muscle cells • cardiac muscle cells cardiac muscle cells • liver cells. liver cells.

Bone marrow stromal cells may differentiate into: cardiac Bone marrow stromal cells may differentiate into: cardiac muscle cells and skeletal muscle cells.muscle cells and skeletal muscle cells.

Brain stem cells may differentiate into: blood cells and skeletal Brain stem cells may differentiate into: blood cells and skeletal

muscle cells. muscle cells.


What are embryonic stem cells?What are embryonic stem cells?

Aare derived from embryos originated from eggs Aare derived from embryos originated from eggs that have been fertilized in vitro in an that have been fertilized in vitro in an in vitroin vitro fertilization clinic and donated to research fertilization clinic and donated to research institutions with donors consent. institutions with donors consent.

The The embryosembryos from which from which human embryonic human embryonic stem cellsstem cells are derived are typically are derived are typically four or four or five days oldfive days old and are a hollow microscopic ball and are a hollow microscopic ball of cells called the of cells called the blastocystblastocyst which includes which includes three stages; three stages; • TrophoblastTrophoblast, which is the layer of cells that , which is the layer of cells that

surrounds the blastocystsurrounds the blastocyst• BlastocoelBlastocoel, which is the hollow cavity inside the , which is the hollow cavity inside the

blastocystblastocyst• Inner cell massInner cell mass,, which is a group of approximately which is a group of approximately

30 cells at one end of the blastocoel.30 cells at one end of the blastocoel.


How are embryonic stem cells grown in the How are embryonic stem cells grown in the laboratory?laboratory?

Growing cells in the laboratory is known as Growing cells in the laboratory is known as cell cell cultureculture..

Human embryonic stem cells are isolated by Human embryonic stem cells are isolated by

transferring the transferring the inner cell massinner cell mass into a plastic into a plastic laboratory culture dish that contains a nutrient broth laboratory culture dish that contains a nutrient broth known as known as culture mediumculture medium. .

The inner surface of the culture dish is typically The inner surface of the culture dish is typically coated with mouse embryonic skin cells that have coated with mouse embryonic skin cells that have been treated so they will not divide. This coating been treated so they will not divide. This coating layer of cells is called a layer of cells is called a feeder layerfeeder layer. .


The reason for having the mouse cells in the bottom of The reason for having the mouse cells in the bottom of the culture dish is to give the inner cell mass cells the culture dish is to give the inner cell mass cells a a sticky surfacesticky surface to which they can attach. In addition, to which they can attach. In addition, these feeder cells these feeder cells release release nutrientsnutrients into the culture into the culture medium. medium.

Over the course of several days, the ESC proliferate and Over the course of several days, the ESC proliferate and begin to crowd the culture dish. begin to crowd the culture dish. Cells are removed gently and plated into several fresh Cells are removed gently and plated into several fresh culture dishes. The process of replating the cells is culture dishes. The process of replating the cells is repeated many times and for manymonths, and is repeated many times and for manymonths, and is called called subculturingsubculturing. Each cycle of subculturing the . Each cycle of subculturing the cells is referred to as a cells is referred to as a passagepassage. .


Embryonic stem cells that have proliferated Embryonic stem cells that have proliferated in cell culture for six or more months without in cell culture for six or more months without differentiating, are differentiating, are pluripotentpluripotent, and appear , and appear genetically normal and are referred to as an genetically normal and are referred to as an embryonic stem cell lineembryonic stem cell line..

Once cell lines are established, or even Once cell lines are established, or even before that stage, batches of them can be before that stage, batches of them can be frozen and shipped to other laboratories for frozen and shipped to other laboratories for further culture and experimentation.further culture and experimentation.


What laboratory tests are used to identify embryonic What laboratory tests are used to identify embryonic stem cells?stem cells?

At various points during the process of At various points during the process of generating embryonic stem cell lines, generating embryonic stem cell lines, scientists test the cells to see whether they scientists test the cells to see whether they exhibit the fundamental properties that make exhibit the fundamental properties that make them embryonic stem cells.them embryonic stem cells.

These tests includeThese tests include;; • growing and subculturing the stem cells for many growing and subculturing the stem cells for many

months. This ensures that the cells are capable of months. This ensures that the cells are capable of long-term self-renewal. long-term self-renewal.


• Scientists inspect the cultures through a microscope Scientists inspect the cultures through a microscope to see that the cells look healthy and remain to see that the cells look healthy and remain undifferentiatedundifferentiated. .

• Using specific techniques to determine the Using specific techniques to determine the presence of presence of surface markerssurface markers that are found only that are found only on undifferentiated cells. on undifferentiated cells.

• Another important test is for the presence of a Another important test is for the presence of a protein called protein called Oct-4Oct-4,, which is typically made by which is typically made by undifferentiated cells. Oct-4 is a transcription factor, undifferentiated cells. Oct-4 is a transcription factor, helps turn helps turn genesgenes on and off at the right time, which on and off at the right time, which is an important part of the processes of cell is an important part of the processes of cell differentiationdifferentiation and embryonic development. and embryonic development.

• Examining the chromosomes under a microscope. Examining the chromosomes under a microscope. This is a method to assess whether the This is a method to assess whether the chromosomes are damaged or if the number of chromosomes are damaged or if the number of chromosomes has changed. chromosomes has changed.


However, microscopy examination of However, microscopy examination of chromosomes, does chromosomes, does not not detect genetic detect genetic mutations in the cells. mutations in the cells.

Determining whether the cells can be Determining whether the cells can be subcultured after freezing, thawing, and subcultured after freezing, thawing, and replating. replating.

Testing whether the human embryonic stem Testing whether the human embryonic stem cells are pluripotent by;cells are pluripotent by; • allowing the cells to differentiate spontaneously in allowing the cells to differentiate spontaneously in

cell culturecell culture• manipulating the cells so they will differentiate to manipulating the cells so they will differentiate to

form specific cell typesform specific cell types• injecting the cells into an immunosuppressed injecting the cells into an immunosuppressed

mouse to test for the formation of a benign tumor mouse to test for the formation of a benign tumor called a called a teratomateratoma. .


Teratomas typically contain a mixture of many Teratomas typically contain a mixture of many differentiated or partly differentiated cell typesdifferentiated or partly differentiated cell typesThis is an indication that the embryonic stem This is an indication that the embryonic stem cells are capable of differentiating into multiple cells are capable of differentiating into multiple cell types.cell types.


How are embryonic stem cells are stimulated to How are embryonic stem cells are stimulated to differentiate?differentiate?

As long as the embryonic stem cells in culture are As long as the embryonic stem cells in culture are grown under certain conditions, they can remain grown under certain conditions, they can remain undifferentiated (unspecialized). But if cells are undifferentiated (unspecialized). But if cells are allowed to clump together to form allowed to clump together to form embryoid embryoid bodiesbodies, they begin to differentiate spontaneously., they begin to differentiate spontaneously.

Although spontaneous differentiation is a good Although spontaneous differentiation is a good indication that a culture of embryonic stem cells is indication that a culture of embryonic stem cells is healthy, it is not an efficient way to produce healthy, it is not an efficient way to produce cultures of specific cell types.cultures of specific cell types.


To generate cultures of specific types of differentiated To generate cultures of specific types of differentiated cells—heart muscle cells, blood cells, or nerve cells, cells—heart muscle cells, blood cells, or nerve cells, for example—scientists try to control differentiation of for example—scientists try to control differentiation of embryonic stem cells by; embryonic stem cells by;

• Change the chemical composition of the culture medium Change the chemical composition of the culture medium • Alter the surface of the culture dish Alter the surface of the culture dish • Modify the cells by inserting specific genesModify the cells by inserting specific genes

If scientists can reliably direct the differentiation of If scientists can reliably direct the differentiation of embryonic stem cells into specific cell types, they embryonic stem cells into specific cell types, they may be able to use the resulting, differentiated cells may be able to use the resulting, differentiated cells to treat certain diseases at some point in the future.to treat certain diseases at some point in the future.


Diseases that might be treated by transplanting cells Diseases that might be treated by transplanting cells generated from human embryonic stem cells include generated from human embryonic stem cells include Parkinson's disease, diabetes, traumatic spinal cord Parkinson's disease, diabetes, traumatic spinal cord injury, Purkinje cell degeneration, Duchenne's injury, Purkinje cell degeneration, Duchenne's muscular dystrophy, heart disease, and vision and muscular dystrophy, heart disease, and vision and hearing loss.hearing loss.


What are the similarities and differences What are the similarities and differences between embryonic and adult stem cells?between embryonic and adult stem cells?

Adult and embryonic stem cells differ in the Adult and embryonic stem cells differ in the numbernumber and and typetype of differentiated cells types they can become. of differentiated cells types they can become.

Embryonic stem cellsEmbryonic stem cells can become all cell types of the can become all cell types of the body because they are body because they are pluripotentpluripotent. . Adult stem cells are generally limited to differentiatingAdult stem cells are generally limited to differentiating intodifferent cell types of their tissue of origin. However, intodifferent cell types of their tissue of origin. However, some evidence suggests that adult stem cell some evidence suggests that adult stem cell plasticityplasticity may exist, increasing the number of cell types a given may exist, increasing the number of cell types a given adult stem cell can become.adult stem cell can become.


Large numbers of embryonic stem cells can be Large numbers of embryonic stem cells can be relatively easily grown in culture, while adult stem relatively easily grown in culture, while adult stem cells are rare in mature tissues and methods for cells are rare in mature tissues and methods for expanding their numbers in expanding their numbers in cell culturecell culture have not have not yet been worked out. This is an important yet been worked out. This is an important distinction, as large numbers of cells are needed for distinction, as large numbers of cells are needed for stem cell replacement therapies.stem cell replacement therapies.

A potential advantage of using stem cells from an A potential advantage of using stem cells from an adult is that the patient's own cells could be adult is that the patient's own cells could be expanded in culture and then expanded in culture and then reintroducedreintroduced into the into the patient. The use of the patient's own adult stem cells patient. The use of the patient's own adult stem cells would mean that the cells would not be rejected by would mean that the cells would not be rejected by the immune system. the immune system.

Embryonic stem cells from a donor introduced into a Embryonic stem cells from a donor introduced into a patient could cause patient could cause transplant rejectiontransplant rejection. .


What are the potential uses of human stem What are the potential uses of human stem cells?cells?

What are the obstacles that must be overcome What are the obstacles that must be overcome before these potential uses will be realized?before these potential uses will be realized?

There are many ways in which human stem There are many ways in which human stem cells can be used in cells can be used in basicbasic research and in research and in clinical clinical research. However, there are many research. However, there are many technical hurdles between the promise of technical hurdles between the promise of stem cells and the realization of these uses, stem cells and the realization of these uses, which will only be overcome by continued which will only be overcome by continued intensive stem cell research.intensive stem cell research.


Potential usesPotential uses

Studies of Studies of human embryonic stem cellshuman embryonic stem cells may yield may yield information about the complex events that occur information about the complex events that occur during human during human developmentdevelopment. A primary goal of this . A primary goal of this work is to identify how work is to identify how undifferentiatedundifferentiated stem cells stem cells become differentiated. Scientists know that turning become differentiated. Scientists know that turning genesgenes on and off is central to this process. Some of on and off is central to this process. Some of the most serious medical conditions, such as cancer the most serious medical conditions, such as cancer and birth defects, are due to abnormal and birth defects, are due to abnormal cell divisioncell division and and differentiationdifferentiation. .

A better understanding of the genetic and molecular A better understanding of the genetic and molecular controls of these processes may yield information controls of these processes may yield information about how such diseases arise and suggest new about how such diseases arise and suggest new strategies for therapy. strategies for therapy.


Human Human stem cells could also be used to test new drugs. For stem cells could also be used to test new drugs. For example, new medications could be tested for safety on example, new medications could be tested for safety on differentiated cells generated from human differentiated cells generated from human pluripotentpluripotent cell lines. cell lines. Other kinds of cell lines are already used in this way. Other kinds of cell lines are already used in this way. Cancer cell lines, for example, are used to screen Cancer cell lines, for example, are used to screen potential anti-tumor drugs. But, the availability of potential anti-tumor drugs. But, the availability of pluripotent stem cells would allow drug testing in a pluripotent stem cells would allow drug testing in a wider range of cell types.wider range of cell types.


Perhaps the most important potential application of Perhaps the most important potential application of human stem cells is the generation of cells and human stem cells is the generation of cells and tissues that could be used for tissues that could be used for cell-based cell-based therapiestherapies. .

Today, donated organs and tissues are often used to Today, donated organs and tissues are often used to replace ailing (ill) or destroyed tissue, but the need replace ailing (ill) or destroyed tissue, but the need for transplantable tissues and organs far outweighs for transplantable tissues and organs far outweighs the available supply. the available supply.

Stem cells, directed to differentiate into specific cell Stem cells, directed to differentiate into specific cell types, offer the possibility of a renewable source of types, offer the possibility of a renewable source of replacement cells and tissues to treat diseases replacement cells and tissues to treat diseases including including Parkinson's and Alzheimer's diseases, Parkinson's and Alzheimer's diseases, spinal cord injury, stroke, burns, heart disease, spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis, diabetes, osteoarthritis, andand rheumatoid arthritis rheumatoid arthritis..


For exampleFor example, it may become possible to generate , it may become possible to generate healthy heart muscle cells in the laboratory and then healthy heart muscle cells in the laboratory and then transplant those cells into patients with chronic hearttransplant those cells into patients with chronic heart disease. disease. Preliminary research in mice and other animals indicates Preliminary research in mice and other animals indicates that bone marrow stem cells, transplanted into a that bone marrow stem cells, transplanted into a damaged heart, can generate heart muscle cells and damaged heart, can generate heart muscle cells and successfully repopulate the heart tissue. Other recent successfully repopulate the heart tissue. Other recent studies in studies in cell culturecell culture systems indicate that it may be systems indicate that it may be possible to direct the possible to direct the differentiationdifferentiation of embryonic stem of embryonic stem cells or adult bone marrow cells into heart muscle cells (cells or adult bone marrow cells into heart muscle cells (Figure 4).).


In people who suffer from type I diabetes, the cells of In people who suffer from type I diabetes, the cells of the pancreas that normally produce insulin are the pancreas that normally produce insulin are destroyed by the patient's own immune system. New destroyed by the patient's own immune system. New studies indicate that it may be possible to direct the studies indicate that it may be possible to direct the differentiation of human embryonic stem cells in cell differentiation of human embryonic stem cells in cell culture to form insulin-producing cells that eventually culture to form insulin-producing cells that eventually could be used in transplantation therapy for could be used in transplantation therapy for diabetics.diabetics.


Figure 4. Figure 4.

Heart muscle repair with Heart muscle repair with adult stem cellsadult stem cells..


Also, to avoid the problem of immune rejection, Also, to avoid the problem of immune rejection, scientists are experimenting with different scientists are experimenting with different research strategies to generate tissues that research strategies to generate tissues that will not be rejected.will not be rejected.


Thank youThank you

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