Regenerative Medicine

HOW TO HELP THE BODY HEAL ITSELF

Replace
‐ implant new organ

Repair

Regenerate

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 HOW TO HELP THE BODY HEAL ITSELF

Replace

Repair

Regenerate

HOW TO HELP THE BODY HEAL ITSELF

Replace

Repair
‐ add new cells to organ

Regenerate

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 HOW TO HELP THE BODY HEAL ITSELF

Replace

Repair

Regenerate
‐ stimulate cell renewal

Tissue and Organ Transplants

• Classifications of transplant materials:
1. Autogenic (same individual)
• no immune response
• certain procedures only (e.g. ligament repair)
2. Allogenic (same species, different individuals)
• most common (e.g. organ donation)
• can elicit an immunological response
• chronic use of immunological suppressants
3. Xenogenic (different species)
• least common (e.g. porcine aortic valves)
• greatest potential to elicit an immunological response
• must be chemically treated prior to implantation

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 Need for New Therapies

• The clinical need for tissue and organ

replacements rapidly increases each year

• Estimated cost of these transplantations is about

$400 billion annually in the USA alone

What are stem cells

• The basic unit of biological life is the cell.

• All biological life is cellular.

• The specialized cells in the body are

derived from stem cells.

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• Stem cells have two defining attributes:
• The capacity for self‐renewal
• The ability to differentiate into many many different
cell types
• There are about six classes of stem cells. We will
discuss the two most important classes of stem
cells:
• Embryonic stem cells
• Adult stem cells

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 Embryonic Stem Cells (ESC)

Embryonic Stem Cells (ESC)

• Derived from the inner cell mass of a blastocyst

• A blastocyst is a hollow ball of cells formed 4‐6
days after a human egg is fertilized

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 Embryonic Stem Cells and Embryonic
Stem Cell Lines
• Cell lines are from one separated cells and the
daughter cells are alike and grow indefinitely.

Adult Stem Cells

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 Adult Stem Cells

• Many adult tissues have stem cells.

• The most well studied are the blood stem cell
(hematopoietic stem cell or HSC used in bone
marrow transplants) and the neural stem cell
• Recently, it was discovered that an adult stem cell
from one tissue may act as a stem cell for another
tissue, i.e. blood to neural

Stem Cells and Regenerative Medicine

• With stem cell therapy (embryonic or adult),

there is enormous promise of treating diseases
previously thought to be unmanageable
• The question is not whether or not to use stem
cells. The question is whether to use adult or
embryonic stem cells

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• Potential Applications of Stem Cells
• Using stem cells to make white blood cells is becoming
an effective way to treat leukemia
• Stem cells from umbilical cord blood used to treat
sickle cell anemia and other blood deficiencies
• Stem cells from fat have been used to form bone tissue
in the human skull
• Repair of heart cells
• Adult stem cells isolated from brain and used to make
neurons in culture

• Cloning
• Refers to making a copy of something
• Reproductive cloning intent is to create a baby
• Therapeutic cloning provides stem cells that
are a genetic match to a patient who requires
a transplant
• No fear of immune rejection

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 Human Cloning
Therapeutic vs. reproductive cloning
• How are embryonic stem cells technically related
to cloning ?
• The two issues are related or not related based on the
answer to the following question: “Where did the
nucleus come from in the fertilized egg used to make
the embryonic stem cell.”

Fertilization vs. cloning

• Fertilization of the egg by the sperm brings
together two different sets of DNA to encode for
a unique individual

• Somatic cell nuclear transfer does not

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 Cellular Therapies

• Cellular therapies have the promise to become major

therapeutic modalities of the next century.
• But, cellular therapy is not a new concept:
• blood transfusions routinely performed for several
decades
• Bone marrow transplantation (currently performed)

Bone Marrow Transplantation

• Bone marrow is comprised of 500‐1000 billion

cells and is the most prolific tissue in the body:
• produces ~ 400 billion myeloid cells/day
• regenerates every 2‐3 days
• all of which originate
from a small number of stem cells

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 Gene Therapy

• Genes
• carried on chromosomes; the basic physical and
functional units of heredity
• specific sequences of bases that encode how to
make proteins
• when altered, encoded proteins are unable to
carry out their normal functions, genetic disorders
can result

Gene Therapy

• Gene Therapy is the technique for correcting

defective genes responsible for disease
development.
• How are genes delivered?
• How can genes be sent to the proper tissues and
organs?
• Can it be effective and safe?

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 How is it done?

• Ex vivo gene therapy

• Cells are removed from the patient, treated with
techniques similar to transformation, and then
reintroduced to the person
• In vivo gene therapy
• Introducing genes directly into tissues and organs in
the body
• Challenge is delivering genes only to intended
tissues and not tissues throughout the body

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 Gene Therapy

• In most gene therapy studies, a "normal" gene is

inserted into the genome to replace an "abnormal,"
disease‐causing gene.
• A carrier molecule (vector) must be used to deliver
the therapeutic gene to the patient's target cells.
• Currently, the most common vectors used are
viruses which have been genetically altered to carry
normal human DNA.

• Vectors for Gene Delivery

• Rely on viruses as vectors
• Use viral genome to carry a therapeutic gene or
genes and use virus itself to infect human cells,
introducing the gene
• Adenovirus (common cold)
• Influenza virus (flu)
• Herpes virus (cold sores, some cause STD)
• Must make sure the virus has been genetically
engineered so that it can neither produce
disease nor spread throughout the body

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• Viral Infection of Human Cells
• Bind to and enter cells; release genetic material
(usually DNA) into nucleus or cytoplasm
• Human cell now acts as a host to reproduce the
viral genome and to produce viral RNA and
proteins
• Make Good Vectors
• Efficient at infecting many types of human cells
• Retroviruses (HIV) permanently insert their DNA
into host cell genome
• Some viruses infect only certain types of cells –
good for targeted gene therapy

Gene Therapy Vectors

• Some of the different types of viruses currently
under investigation for use as gene therapy
vectors:
• Adenoviruses
• Retroviruses
• Adeno‐Associated Viruses (AAV)

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 Problems with Gene Therapy
• Short‐Lived Nature
• Problems with the stability of therapeutic DNA once in
the genome and the rapidly dividing nature of many
cells hinder achieving long‐term benefits of gene
therapy. Patients will have to undergo multiple rounds
of gene therapy.
• Immune Response
• There is a risk of stimulating the immune system when
using viral gene delivery vectors, thereby reducing
effectiveness.

Problems with Gene Therapy

• Viral Vectors
• Viruses present a variety of potential problems to the
patient: toxicity, immune and inflammatory responses,
gene control and targeting issues. Also, there is the fear
that the viral vector, once inside the patient, may
recover its ability to cause disease.

• Multi‐Gene Disorders
• Conditions arising from mutations in a single gene are
the best candidates. However, some common disorders
(Alzheimer's, arthritis, diabetes, etc.) are caused by
combined effects of variations in many genes making
them difficult to treat.

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 Tissue Engineering

Tissues

• Tissue is a cellular organizational level intermediate

between cells and a complete organism.
• Organs are then formed by the functional grouping
together of multiple tissues..
• The classical tools for studying tissues are the paraffin
block in which tissue is embedded and then sectioned,
the histological stain, and the optical microscope

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Several types of tissue form an organ

Types of Tissues

• Animal tissues
• Connective
• Muscle
• Nervous
• Epithelial

• Plant tissues
• Epidermis
• Vascular
• Ground

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Animal Tissues

Connective tissues

Muscle tissues

Animal Tissues

Nervous tissue

Epithelial tissues

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 What is Tissue Engineering ?

• Tissue Engineering is the in vitro development

(growth) of tissues or organs to replace or
support damaged or diseased tissues
• Process
• Design a framework or scaffold
• Seed the scaffold with human cells
• Bathe in nutrient‐rich media
• Cells will build layers and assume the shape of the
scaffold

What is Tissue Engineering ?

• Sheets of skin grafts

• 1990s Dr. Charles Vacanti revealed a mouse with
an engineered ear growing on its back
• Seeded with cells from a cow
• Just the outer ear without the inner ear structures that
actually detect sound
• Human bladders, rudimentary kidney

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 Traditional Tissue Sources

• Tissue engineering emerged around 1988

• Interdisciplinary field for the development of
biological substitutes for the repair or regeneration
of tissue of organ function
• Tissue engineering is a potential alternative to
tissue or organ transplantation
• Traditionally required tissue has been transferred
from one place to another

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 Tissue Engineering

• General Paradigm

Scaffolds

Engineered Tissue Concepts

• Start with some building material (e.g.. extracellular

matrix or biodegradable polymer), shape it as
needed.
• Seed it with living cells
• Bathe it with growth factors
• When the cells multiply, they fill up the scaffold and
grow into three‐dimensional tissue.

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 Engineered Tissue Concepts

• Once implanted in the body, the cells recreate their

intended tissue functions
• Blood vessels attach themselves to the new tissue,
the scaffold dissolved, and the newly grown tissue
eventually blends in with its surroundings

Elements of Tissue Engineering

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 Scaffolds

• Control of porosity (diffusion vs. strength), pore size

distribution and interconnectivity.
• Control of rate of degradation (if biodegradable).
• Consistent processing technologies.

Tissue Engineering Scaffolds

Scaffold Materials
•Porous three‐dimensional
•Natural versus synthetic polymers:
• synthetic polymers
• poly(lactide) ,poly(lactide‐co‐glycolide), poly(caprolactone)….
• foams, hydrogels, fibres, thin films

• natural polymers
• collagen, elastin, fibrin, chitosan, alginate….
• fibres, hydrogels

• ceramic
• calcium phosphate based for bone tissue engineering
• porous structures

• permanent versus resortable

• degradation typically by hydrolysis (except for natural materials)
• must match degradation rate with tissue growth

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 Scaffolds

• Various textures and materials

• Encourage cells to grow
• Allow nutrients to permeate
• Won’t harm the patient

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 Culturing of Cells
•Types of Cell Culture
• monolayer (adherent cells)
• suspension (non‐adherent cells)
• three‐dimensional (scaffolds or templates)

Bioreactor

• A bioreactor is a vessel in which is carried out a

chemical process which involves organisms or
biochemically active substances derived from
such organisms.

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 Bioreactor

• A closed system that

continuously supplies:
• Nutrients and oxygen.
• Mechanical stresses.
• Efficient mass transfer
to the growing tissue.

Bioreactors

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 Research is presently being conducted on
several different types of tissues and organs,
including:
 Skin
 Cartilage
 Blood Vessels
 Bone
 Muscle
 Nerves
 Liver
 Kidney
 etc. etc. etc.

Tissue‐engineered products contain mixtures

of the following:

• Biological components—cells
• Can be genetically modified to behave a specific way
• Chemicals
• that tell the tissue to regenerate
• A non‐biological component
• Polymer scaffold
• Fibers, plastic, other natural components
• Gels

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 Transplants that match the patient

• Remove cells from patient

• Grow in culture with or without biomaterials
• Give appropriate chemicals to make cells do what is
needed
• Replace into patient

A new bladder
• To make a bladder one need a
scaffold and several different
type of cells

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 Bone repair
• Obtain patient’s bone
cells
• From the hip
• Seed onto scaffold
• Implant scaffold
• Use body as incubator
• All appropriate growth
factors available
• Remove and implant in
proper location

Using adult stem cells

• Inject into site of

damage
• heart damage
• Muscular dystrophy
• Texture, stiffness and
chemicals in
surroundings may
influence injected cells

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 Muscular Dystrophy
• A genetic disorder that
affects 1 in 3,500 males
• Patients have trouble
walking beginning in
preschool and eventually
die in their 20s due to
weakened heart and lung
muscles
• Dogs with MD were given
injections of healthy cells
and were able to walk
faster and even jump
• Best results came by using
stem cells from healthy
dogs

Polio

• A virus attacks cells in the

spinal cord
• Signal no longer sent to
muscles in the leg
• Muscle wasting occurs
• Stem cell treatment could
• encourage new spinal
neurons to grow
• help new muscle to grow

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 Replacement cells for diabetes
In vivo Islet of Langerhans in pancreas

Engineering bone grafts

• Change stem cells into

bone cells
• with proper growth factors
in cell culture media
• This scaffold can’t be too
big or the cells inside will
die since they will not get
enough oxygen

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 Replacement cartilage

A close up image of cells in the replacement cartilage

• Mimic usual cartilage

environment
• Cartilage cells
• Collagenous scaffold
• Doesn’t require extensive blood
supply

Artificial skin for ulcers and burns

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 Artificial Vessels

• Usually for by‐pass

operation
http://popularmechanics.com/popmec
h/sci/tech/9805TUMDOM.html

Tissue Engineering

• Most successes have been limited to a vascular or thin

tissues (< 200 mm)
• skin, cartilage, cornea

• The most important problems associated with thicker or

more complex tissues include:

•the need for multiple cell types

•the need for the tissue to become vascularized
• vascularization of the 3‐D construct is a critical and
unresolved problem

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 Relative Microbe Sizes

Microorganisms:

• Good
• Decompose organic waste
• Are producers in the ecosystem by photosynthesis
• Produce industrial chemicals such as ethyl alcohol and
acetone
• Produce fermented foods such as vinegar, cheese, and
bread
• Bad
• Pathenogenic

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 Ubiquity of Microorganisms
• Found nearly everywhere!!
• Occur in large numbers
• Live in places many other organisms cannot

Microbes & Industry

Industry: Fermentation products (ethanol, acetone, etc.)
Food: Wine, cheese, yogurt, bread, half‐sour pickles, etc.
Biotech: Recombinant products (e.g., human insulin, vaccines)
Environment: Bioremediation

Each carton of Bugs+Plus provides easy to follow

step‐by‐step instructions, containers of
specially‐formulated wet and dry nutrients and
a container of microbes cultured for their ability
to digest oil and other petroleum derivatives.

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 Microbes & Disease

 Microbes both cause and prevent diseases

for latter: antibiotics are made by microbes and normal flora
interfere with pathogen replication
 Microbes produce antibiotics used to treat diseases
 The single most important achievement of modern
medicine is the ability to treat or prevent microbial
disease

Microorganisms:

Fungi

Protozoa Algae Viruses

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 Normal flora

• Normal flora are bacteria, fungi, and protozoa

that live on or within the bodies of animals and
plants.
• Normal flora, by definition, do not cause disease
in healthy individuals. Instead, they are
commensalisms or mutualisms with regard to the
host.
• That is, in addition to basically not harming the
host, they can even do some good.

The Cell Envelope of Bacteria

• Majority of bacteria have a cell envelope

• Lies outside of the cytoplasm
• Composed of two or three basic layers
• Cell wall
• Cell membrane
• In some bacteria, the outer membrane

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 Differences in Cell Envelope Structure

• The differences between gram‐positive and

gram‐negative bacteria lie in the cell envelope
• Gram‐positive
• Two layers
• Cell wall and cytoplasmic membrane
• Gram‐negative
• Three layers
• Outer membrane, cell wall, and cytoplasmic
membrane

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 Viruses

• Viruses are not really living.

• Viruses are obligate intracellular parasites that
can be maintained only inside living cells.
• They cannot multiply outside a living cell, they
can only replicate inside of a specific host.

Viruses types by envelope

• Naked (Capsid )
• i.e. no envelope, easy for the immune system
to kill.
• Enveloped

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Figure 6.10

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 Steps of Virus Replication
1. Adsorption (attachment)
2. Penetration (nucleic‐acid release)
3. Synthesis (of RNA and proteins, as well as
DNA if DNA genome)
4. Maturation (assembly of virion)
5. Release (lysis or chronic release, e.g., budding,
with the latter coinciding with release for
various enveloped viruses)
It is important to realize that variation among viruses is between
virus strains/species; any one kind of virus cannot replicate in
multiple ways, have more than one virion morphology, or vary in
genome type, etc.

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 The Lytic vs. Lysogenic Cycle

Virus Infection
• Viruses infect all sorts of cells, from bacteria to
human cells, but for the most part tend to be host
specific.
• Viruses cause a variety of diseases among all
groups of living things.
• The viral diseases we see are due to the effects of
this interaction between the virus and its host cell
(and/or the host’s response to this interaction).

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 • Viral diseases in humans are controlled by
• preventing transmission
• administering vaccines
• recently by the administration of antiviral drugs.

Antibiotics

• Any viral disease, the major treatment efforts focus

on treatment of symptoms, not removal of the viral
cause.
• Antibiotics are not effective against viruses.
• Antibiotics do not cure viral infections because
viruses use enzymes produced by the host cell,
rather than produce not their own.

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 Vaccines

• Vaccination offers protection for uninfected

individuals.
• Vaccines are made from killed pathogens or
weakened strains that cause antibody production
but not the disease.
• Vaccines are substances that stimulate an immune
response without causing the illness.
• A few antiviral drugs are available that interfere with
viral replication without interfering with host
metabolism in cells free of the virus.

• The immune system can develop long‐term

immunity to some diseases.
• By using this to develop vaccines, which produce
induced immunity.
• Active immunity develops after an illness or vaccine.
Vaccines are weakened (or killed) viruses or bacteria
that prompt the development of antibodies.
Application of biotechnology allows development of
vaccines that are the protein (antigen) which in no
way can cause the disease.
• Passive immunity is the type of immunity when the
individual is given antibodies to combat a specific
disease. Passive immunity is short‐lived.

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