bionic human 7 questions
qqq823
chapter_11.pdf
Stem Cells and
Regenerative Medicine
Chapter 11
Learning Objectives
Identify the differences between the various
types of Stem Cells
Identify the Sources of Stem Cells
Know how Stem Cells can be used in
regenerative medicine and tissue engineering
Through understanding how
these animals regenerate
limbs we can develop an
understanding of why we are
not able to do so, and
potentially learn to overcome
those “roadblocks” to
regeneration.
Salamander -
Epimorphosis
The Salamander’s Trick
The “blastema cells” (shown in light blue) are essentially stem
cells that arose from the normal limb cells “de-differentiating” or
returning to a primitive embryonic-like state.
The salamander’s trick is that it can make it’s cells “go back in
time”, something that human cells are not believed to be capable
of doing.
The basic concept of a stem cell
1. A stem cell can make copies of itself.
2. A stem cell can differentiate, i.e. become a
specialized type of cell. Differentiation is a
series of steps from initial commitment to
becoming a functional, specialized cell.
http://www.stemcellsforhope.com/Stem%20Cell%20Therapy.htm
Characteristics of Stem Cells
Some Definitions
• Somatic Cell: a mature, differentiated cell, i.e. a
skin cell.
• Differentiated (cell): committed to being a
specialized cell.
• Undifferentiated (cell): retains the potential to
become multiple specialized cell types.
• Stem Cell: primitive, undifferentiated cells
– Self-renewing: can give rise to copies of itself.
– Multipotent: can give rise to multiple (but not all) cell
types.
– Pluripotent: can give rise to all cell types of the body.
Some Definitions
• Regeneration: re-growth of lost or damaged
cells, tissues or organs.
• Regenerative Medicine: process of
replacing or regenerating human cells,
tissues or organs to restore or establish
normal function through biomedical
interventions that may involve the use of
stem cells.
Cell Identity • All cells in the body have an identical genotype
(genetic makeup), i.e. the same set of genes.
21,000 protein-coding genes.
• Why do cells have different phenotypes
(observable characteristics)? What makes a liver
cell a liver cell? A brain cell a brain cell?
• When a gene is “expressed” the result is the
production of the protein it codes for.
– Muscle cells produce myosin which drives
contraction.
– Pancreatic beta cells produce insulin.
Cell Identity
• Genes are turned “on” or “off”, and depending
on the combination a cell will take on certain
functional characteristics.
• Differential gene expression determines cell
identity (or phenotype) and is controlled by
signals in the cell’s environment.
This is the point of intervention for the
biomedical scientist. We manipulate the
environment to coax a stem cell into thinking it
needs to become the desired type of cell.
1 genotype, 2 phenotypes
The caterpillar and the butterfly are the same
organism. There is only one set of genes, or genotype,
yet there are 2 wildly different forms, or phenotypes,
which can emerge.
Important for understanding diversity of life and also
for understanding how to control the behavior of cells.
Stem Cell Repair Damaged
Heart • http://www.cbsnews.com/videos/heart-
patient-sees-results-in-stem-cell-study
Classes of Stem Cells
• Embryonic Stem Cells - the infamously debated
cells. Embryonic stem cells are pluripotent,
which means they can give rise to ALL cell types
of the body.
– They are derived from the blastocyst of a
developing embryo.
Classes of Adult Stem Cells
– Hematopoietic Stem Cells: derived from
bone marrow, can give rise to all of the
blood cells and immune system cells.
– Mesenchymal Stem Cells: derived
primarily from bone marrow, but also from
fat tissue (peripheral vs visceral), can give
rise to multiple cell types – but not all (see
next slides).
– The most abundant reservoir of stem cells
in an adult human is the bone marrow.
Classes of Adult Stem Cells
– Stem cells have been found in most adult
organs (brain, heart, lung) however they are
much lower in number & therefore difficult
to harvest and potentially use for therapy.
These cells can give rise to the tissue from
the organ in which they are found.
• Cord Blood Stem Cells – essentially blood
stem cells, with some evidence of potential
for other tissues.
Hematopoietic (Adult) Stem Cells
http://www.allthingsstemcell.com/category/hematopoietic-stem-cells/
Used to treat blood and immune system disorders, as well as to
replenish the bone marrow following chemotherapy in cancer patients.
Cord Blood Stem Cells • Cord blood stem cells are believed to be largely hematopoietic in
nature, although they have also been shown the possibility of
forming other types of cells such as nerve or liver cells.
• Big advantage is that each person could have a “bank” of their
own stem cells to use later in life when diseases arise.
Cord blood stem cells turning
into nerve cells.
Cord blood at the time of
harvest from an infant.
Mesenchymal (Adult) Stem Cells
The idea of (adult) stem cell therapy
for bone and cartilage repair
• Autologous cells – the
patient’s own.
• Bone marrow-derived
mesenchymal stem cells
are harvested from the
marrow of the pelvic bone.
• Cells are differentiated into bone or
cartilage cells “in vitro”, i.e. outside
of the body in the laboratory.
• New bone or cartilage cells are
used for therapy. http://www.miamiherald.com/living/health-fitness/article68194662.html
Embryonic Stem Cells
• Obtained from embryo 4-5 days after
fertilization.
• These stem cells are “pluripotent”, able to
differentiate into the 220 cell types of the
body.
• Embryonic stem cells can also propagate
themselves indefinitely.
Potential of Embryonic Stem Cells
http://www.stemcellsforhope.com/images/StemPic1-small.JPG
Safety Issues with
Embryonic Stem Cells • Teratoma: Malignant teratoma is a type of cancer made of cysts
that contain one or more of the three layers of cells found in a
developing fetus at the gastrula stage. These layers are called
ectoderm, mesoderm, and endoderm.
https://www.youtube.com/w
atch?v=kTHoTMr_0U8
Safety Issues with Embryonic
Stem Cells • If un-differentiated embryonic stem cells
are injected into an animal they form
teratomas.
– Therefore they must be differentiated into the
desired cell type prior to therapeutic use.
– This implies that precise purification is
needed to remove any lingering
undifferentiated cells. Even one cell could
lead to the formation of a teratoma.
Alternative sources of
pluripotent stem cells
• There are 2 additional ways to produce
pluripotent cells that do not involve
fertilization of an egg.
1. Therapeutic cloning (has never happened in humans). Requires an egg cell and the
nucleus of a normal adult cell.
2. Induced pluripotency (has been done in humans) – involves genetic manipulation of
an adult somatic cell (a cell forming the body
of an organism).
Induced pluripotent stem cells
• Very new technology
• Utilizes a gene therapy approach
to force a normal, somatic cell (a
differentiated cell of the body) to
express 4 particular genes (only
4!!!) that “reprogram” the somatic
cell into an induced pluripotent
stem cell.
"Shinya yamanaka10" by National Institutes of
Health -
http://nihrecord.od.nih.gov/newsletters/2010/02_19_
2010/story1.htm. Licensed under Public Domain via
Commons -
https://commons.wikimedia.org/wiki/File:Shinya_ya
manaka10.jpg#/media/File:Shinya_yamanaka10.jpg
Shinya Yamanaka (山中 伸弥) The 2012 Nobel Prize in
Physiology "for the discovery
that mature cells can be
reprogrammed to become
pluripotent”.
Stimulus-triggered acquisition of
pluripotency (STAP) • STAP - an alleged
method of generating
pluripotent stem cells
Haruko Obokata (小保方 晴子)
She and her colleagues had demonstrated a
surprisingly simple way of turning ordinary
body cells – she used mouse blood cells –
into something very much like embryonic
stem cells. All you need to do is drop them
into a weak bath of citric acid, let them soak
for half an hour.
Nobody can reproduce her result,
including herself
Obokata probably had falsified
and fabricated data
STAP cells were actually
embryonic stem cells, and the
mixup was probably not
accidental
On August 5, 2014, Obokata's
mentor and co-author Yoshiki
Sasai committed suicide
Obokata, Haruko;
Wakayama, Teruhiko;
Sasai, Yoshiki; et al.
(2014). "Stimulus-
triggered fate conversion
of somatic cells into
pluripotency". Nature 505
(7485): 641–647.
Everyday Stem Cells:
Regeneration of Intestinal Lining
The mechanism of intestinal
regeneration (new surface
every 2-3 days) involves
stem cells which reside in a
“niche”. They are constantly
dividing to make more stem
cells and also enough cells
to reform the intestinal lining.
Liver Regeneration • A (healthy) liver will completely regenerate following
surgical removal of up to 2/3 of the liver mass (liver
cells only live about 150 days).
• This regenerative process is driven by proliferation,
i.e. multiplication, of pre-existing liver cells, and does
not involve activity of stem cells as in the intestine.
• The problem: With aging and disease (i.e. cirrhosis)
the liver loses it’s ability to regenerate.
Diseases involving cellular death in organs
that do not easily regenerate
• Alzheimer’s, Parkinson’s disease - brain cells
• Retinitis pigmentosa - retinal cells
• Diabetes - pancreatic cells
• Muscular dystrophy – muscle cells
• Heart failure and other cardiovascular
diseases - heart muscle/vascular cells
At the current time the only foreseeable solution to these
problems is replacement of the dead cells with healthy
functional cells derived from stem cells. In many cases
only embryonic stem cells have currently been shown to
form the needed cell type, for example retina and certain
types of brain cells.
Macular Degeneration • A chronic eye disease causing
vision loss in the center of the
field of vision. Dry macular
degeneration caused by
deterioration of macula in the
center of the retina. No
treatments available.
• Wet macular degeneration is
caused by blood vessels growing
under retina leaking blood and
fluid. May be treated with
medication or lasers to destroy
blood vessels.
Clinical trials so far in the U.S. involving the use
of human embryonic stem cells
From the horse’s mouth: https://clinicaltrials.gov/ct2/results?ter
m=human+embryonic+stem+cells&recr
=Open
The first safe, and reportedly effective use of a
human embryonic stem cell-based therapy was for
the treatment of macular degeneration.
https://www.youtube.com/watch?v=ewEdR5_V-v0
The Ethical Debate • At what moment does a life begin?
– A deeply personal opinion
• Left over fertilized eggs from in vitro
fertilization will be discarded.
– Is this a waste?
• One must consider these issues before
deciding whether they support human
embryonic stem cell research.
Therapeutic Cloning: Dolly the Sheep http://nyti.ms/16Xxmq8
Tissue Engineering and Regenerative Medicine
Why Tissue Engineering?
• Examine the challenges of organ transplantation -
(The Clinical Problem!!!)
• Surgical methods are advanced and highly
successful when suitable donor organs are
available, but…
– Limited donor availability – genetic matching;
size matching. Should be a healthy organ.
– Immune rejection (not a long-term solution)
Why Tissue Engineering?
• The idea is to utilize the patients own cells outside
of the body in combination with scaffolds and
bioreactors to create tissues that can be implanted
without the problem of immune rejection.
Why outside the body?
• Remember our cells have the capacity to grow and
regenerate but there are roadblocks to regeneration
in the body, i.e. inflammation. Outside of the body
we have control over the environment and therefore
cell behavior.
Tissue Transplantation
• Autografts – from same organism (no immune rejection) – Middle 1/3rd of patellar tendon graft for ACL reconstruction – Hip bone fragments for bridges in spinal fusion – Coronary vessel bypass – utilize healthy vessels from the lower extremities – Stem cells from the bone marrow!!
• Allografts – from same species (one human to another) – Traditional organ transplants
• Xenografts – tissue from another species (animal to human) – Porcine (pig) heart valves
– Bovine bone for packing defects
– Encapsulated porcine pancreatic islets
Porcine heart valve From Veterinary Transplant Services
Tissue Engineered Heart Valves Autologous Cells + Scaffolds + Bioreactors
Pig heart valves (Gold
Standard):
1. Prepared with
glutaraldehyde, a toxic
fixing agent that gives
the tissue a leathery
strength.
2. They calcify over time.
Biodegradable
scaffold
(PGA polymer)
Seeding of
autologous
cells,
culture in
vitro
Bioreactor
allows for flow
conditions
that simulate
natural heart
valve fluid
forces.
Tissue Engineered Heart Valves (still in the development stages)
Porcine heart valves
(xenografts) calcify over
time, as part of the
immune-inflammatory
response, giving them a
limited life, typically 5-10
years.
Tissue-engineered heart valves
(autografts) tested in animals do
not show long term signs of
calcification.
https://www.youtube.com/watch
?v=7p8VrGr-Drk
Shape?
Example of an Engineered Xenograft: Alginate-encapsulated Pig Islets
for treatment of Diabetes • Alginate is a natural polymer derived from seaweed.
• Tunable pore size: big enough for movement of nutrients
and insulin, but small enough to prevent penetration of
antibodies or immune cells.
http://www.biotechlearn.org.nz/focus_stories/pig_cell_transplants/images/encapsulated_pig_islet_diagram
http://www.voxy.co.nz/national/lct-begins-trials-
implant-pig-tissue-auckland-diabetics/5/18932
Tissue Engineering
Tissue engineering is the
use of a combination of
cells, engineering and
materials methods, and
suitable biochemical and
physical factors to
improve or replace
biological functions.
The Tissue Engineering Paradigm
Implantation
& Integration
3-dimensional
culture
(Scaffolds)
Mechanical
stimulation
Chemical
stimulation
Autologous
Tissue Cells
Patients own
healthy tissue Stem
Cells
Cell
Proliferation
Differentiation
Extracellular
Matrix Production Tissue
Remodeling
Homeostasis
Homeostasis • Homeostasis (homeo = same, stasis = stable) - tendency of tissues
& organs within the body to remain functionally stable
through coordinated interactions with other parts of body.
• In the context of tissue engineering, the way tissues
participate in homeostasis is through the blood, i.e.
vascularization is a central challenge.
Classic example: blood glucose regulation “Engineer’s View” of Homeostasis
Homeostasis
https://www.youtube.com/watch?v=G-nffUdhwjE
The Trinity (+1) of Tissue Engineering
and Regenerative Medicine
1. Cells classes of stem cells
(see previous lecture), or somatic cells, i.e.
epidermal and dermal cells from the patient
for skin grafts. Remember that certain (healthy) tissues such as skin,
liver, and intestine have the capacity to proliferate.
2. Scaffolds
3. Bioactive molecules
--------------------
4. Bioreactors
The idea of a “Scaffold”
• Tissues are composed of cells (bricks) and
extracellular matrix (mortar). Extracellular
matrix provides support and structure, but
should be thought of as a continuum of
fibers within a fluid gel.
• The term "scaffold” in tissue engineering
and regenerative medicine represents an
engineered equivalent of the natural
extracellular matrix.
Scaffold for tissue engineered
heart valve Hybrid valve at various stages of development.
Nitinol structure along with
(A) smooth muscle,
(B) fibroblast/myofibroblast,
(C) endothelial cells
all encapsulated in collagen as the
first, second, and third layers,
respectively.
Polymeric
https://www.researchgate.net/publication/5894568_Prosthetic_heart_valves_Catering_for_the_few/figures?lo=1
http://kheradvar.eng.uci.edu/research.php?cat=hybridvalve
The idea of a “Scaffold”
• Scaffolds have several essential
functions:
– Provides a three-dimensional space for
new tissue development.
– Delivers cells and maintains their
localization at the site of implantation.
– Directs macroscopic size/shape of new
tissue, i.e. if a vascular graft is needed the
scaffold should be a tubular structure with
the appropriate diameter to match.
The Extracellular Matrix (Nature’s Scaffold)
1. Structural support -
acts as a malleable
“mortar” or “glue” that
holds cells together in
tissue.
2. Composition – the most
common components
are collagens (> 20
different types of
collagen proteins).
3. Information - each
tissue’s extracellular
matrix is unique.
Another control element
for the scientist.
Ideal scaffold designs should incorporate all of these
aspects, taking care to match the design parameters as
closely to the target tissue as possible.
Some tissues are primarily
extracellular matrix
• Connective Tissues such
as tendons and ligaments
are primarily composed of
collagen type I fibers.
• Cartilage is mostly
extracellular matrix,
composed of collagen
type II and
glycosaminoglycans
(sugar polymers) with a
very high water content.
The Tissue Engineering Paradigm: Utilize scaffolding materials to arrange cells
in a 3-dimensional structure
http://www.nature.com/nature/journal/v414/n6859/fig_tab/414118a0_F1.html
Cells isolated from patient
and maintained in culture in
vitro to expand population.
Cells seeded in vitro into a
3-D “scaffold” and treated
with various chemical
stimulants to promote
tissue development.
“Tissue construct” is
implanted once cellular
development has been
established.
The scaffold eventually
degrades, being replaced
by remodeled tissue.
Engineering the Scaffolds
Natural or synthetic biomaterials.
Synthetic polymers: e.g. poly(glycolic acid) [PGA], poly(lactic acid) [PLLA]
Natural materials: usually composed of ECM components (e.g., collagen,
elastin, fibrin, or de-cellularized tissues, such as a heart valve free of cells)
---------------------------------------------------------------------------------
Engineering the Scaffolds
The scaffold material should be biocompatible and
satisfy these basic requirements:
1. Favorable cell attachment.
2. Mechanical properties to match the target tissue.
3. Biodegradable - we want the cells to remodel the tissue
and produce their own extracellular matrix.
4. No adverse reactions or toxicity to degradation
products.
-------------------------------------------------------------------------------
• Interconnecting pores are a key design feature of scaffolds to allow for movement of nutrients and cells throughout the structure.
Delivery of oxygen and nutrients (and removal of waste products)
http://www.sciencedirect.com/science/article/pii/S1350946211000279
Capillaries are 25-100 micrometers in all tissues. A cell is
~ 10-20 microns in diameter on average, some much larger.
When attempting to construct 3D tissues outside of body,
scaffold must account for this transport of nutrients and
waste by having an open & interconnected pore structure.
• Angiogenesis – the growth of new blood vessels from pre-existing blood vessels.
• Once an engineered tissue is implanted in the body,
angiogenesis must occur in order for the tissue to
remain viable and participate in homeostasis.
Cancer has evolved mechanisms to promote
angiogenesis – this is a required step for the
tumor to grow and eventually spread.
Tissue Engineers attempt to
grow blood vessels within
tissue in vitro, so they can
“connect’ upon implantation.
https://youtu.be/M4jMLXE-CBc
https://www.youtube.com/watch?v=O5r-T6ANKto
Use preexisting blood vessels
https://www
.sciencedail
y.com/relea
ses/2008/0
1/08011314
2205.htm
http://www.nyt
imes.com/201
2/09/16/healt
h/research/sci
entists-make-
progress-in-
tailor-made-
organs.html
The Trinity (+1) of Tissue Engineering
and Regenerative Medicine
1. Cells classes of stem cells
(see previous lecture), or somatic cells, i.e.
epidermal and dermal cells from the patient
for skin grafts. Remember that certain (healthy) tissues such as skin,
liver, and intestine have the capacity to proliferate.
2. Scaffolds
3. Bioactive molecules
--------------------
4. Bioreactors
Growth Factors (Bioactive Molecules)
typically used in regenerative strategies
http://rsif.royalsocietypublishing.org/content/8/55/153.full.pdf+html
Signals in the cell’s environment: Extracellular Matrix and Growth Factors
http://rsif.royalsocietypublishing.org/content/8/55/153.full.pdf+html
Incorporating growth factors into scaffolds
http://rsif.royalsocietypublishing.org/content/8/55/153.full.pdf+html
Schematic of two
tissue engineering
approaches using
synthetic ECMs to
present growth
factors to tissues.
(a) Physically encapsulated bioactive factors can be released from synthetic
ECMs to target specific cell populations to migrate and direct tissue
regeneration.
(b) Alternatively, growth factors can be chemically bound to the material system,
making them available to cells that infiltrate the material.
The Trinity (+1) of Tissue Engineering
and Regenerative Medicine
1. Cells classes of stem cells
(see previous lecture), or somatic cells, i.e.
epidermal and dermal cells from the patient
for skin grafts. Remember that certain (healthy) tissues such as skin,
liver, and intestine have the capacity to proliferate.
2. Scaffolds
3. Bioactive molecules
--------------------
4. Bioreactors
Bioreactors
• Bioreactor - a manufactured or engineered
device or system that supports a biologically
active environment. In the context of Tissue
Engineering, a bioreactor improves cell growth
and tissue formation in 3-dimensional tissue
constructs.
Bioreactors
• Tissue Engineers utilize bioreactors for 2
general purposes:
– To improve mass transport, i.e. delivery of oxygen and nutrients and the removal of waste
products – this replaces the function of the
vasculature in body. We need to keep tissue alive!!!
– To provide mechanical cues that “condition” the tissue for it’s in vivo function, i.e. flow of liquid
medium through engineered blood vessels,
mechanical stretch of engineered muscle, ventilation
of engineered lungs. Being alive isn’t enough, we
need to “train” the tissue to perform the required
function.
Examples of bioreactors in
tissue engineering
For mechanical stretching of engineered
muscle tissue: https://www.youtube.com/watch?v=XmDeaP6n9vA
For pulsatile flow perfusion of engineered
blood vessels: https://www.youtube.com/watch?v=5jb7ed2iCJs
For a low pulsatile flow that grants the correct
opening and closing of the valve without high
shear stresses: https://www.youtube.com/watch?v=UFZzX-X-DzY
Successful implantation of
tissue engineered bronchus
Successful implantation of a
Tissue Engineered Trachea
http://www.youtube.com/watch?v=_GyQWAiDu0w
Tissue Engineered Bladder (Tengion company)
http://bmb.oxfordjournals.org/content/97/1/81.full
Scaffold without cells New Bladder sutured
in place Wrapped in “fibrin
glue” and omentum.
1. Scaffold composed of PLGA (synthetic)
and collagen (natural) is shaped into the
required size for the patients bladder.
2. Cells isolated from the patients bladder
are cultured on the scaffold for ~ 8 weeks.
3. New “engineered” bladder is implanted
by anastomosis and sutured into place.
4. New bladder is wrapped in fibrin glue
and omentum to enhance vascularization.
The Tissue Engineering and Stem Cell industry
Currently Available Tissue
Engineered “Products”
• Bone grafts: InfuseTM (Medtronic)
• Wound healing grafts/dressings: ApligraftTM (Organogenesis)
• Cell banking services: cord blood
• RESTORETM small intestine submucosa (duPont)
-------------------------------------
Countless therapies (and eventually products/services) are
still in the research and development stages.
This is due to the complexity of organs such as pancreas,
liver, lungs, hearts, eyes, etc
It is impossible to put a timeline, much depends on funding
for basic research – but we are looking at decades,
maybe 10 years for some applications, perhaps 30 years
or longer for others.
http://www.ted.com/talks/anthony_atala_printing_a_human_kidney?language=en
Printing a human kidney
