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Innovation and Development of Study Field
Nanomaterials at the Technical University of Liberec
nano.tul.cz
These materials have been developed within the ESF
project: Innovation and development of study field
Nanomaterials at the Technical University of Liberec
CELLS FOR
TISSUE ENGINEERING
doc. RNDr. Evžen Amler, CSc.
RNDr. Lucie Koláčná, Ph.D.
TISSUE ENGINEERING
 Biopsy (donor-tissue extraction)
CELL ISOLATION AND CULTIVATION
 Scaffolds, seeding and cultivation (3-D)
 Implantation
 Detection – property analysis
CELLS
Appropriate cells – guts of successful therapy
Cells
Source
 tissue specific cells
 tissue
(? sufficiency)
 stem cells
 cell line
CELLS
Cells
 tissue-specific
 stem cells
Sources
 tissue
 cell lines
Properties




non-immunogenic
high proliferation rate
appropriate manipulation
capability of differentiation into different cell types (stem cells)
Proliferation stimulation, differentiation (growth factors)
CELLS
Cell sources
Recipient healthy tissue – autologous cells
application helps avoid complications of immune rejection
Cells isolated from different species
from a genetically unidentical, same species (allogenic)
or from different species (xenogennic)
alternative, risk of complications of immune rejection
Autologous – from the same individual
(tissue engineering: differentiated and progenitor cells)
donor and recipient are the same individual
Allogenic – from member of the same species
different donor and recipient, same species – both e.g. human;
the genes at one or more loci are not identical in sequence in each
organism
Heterologous (xenogennic) – from one species to another
donor and recipient different species, e.g. pigs – humans
STEM CELLS
STEM CELLS
Undifferentiated cells from the embryo, fetus, or adult
that has, under certain conditions, the ability to
reproduce itself (capacity to renew itself)
and to give rise to specialized differentiated cell types
with characteristic properties (formation of tissues and
organs)
Application
 cell therapy
 regenerative and reparative medicine
STEM CELLS
Differentiation – unspecialized cell (such as a stem cell) becomes
specialized into one of the many cells that make up the body –
differentiated cells with specialized function and structure. e.g. liver,
heart, muscle).
Dedifferentiation – differentiated cell reverts to an earlier
developmental stage. Regression of a specialized cell to a simpler,
more embryonic, unspecialized form. Loss of morphology,
biochemical properties and function.
Redifferentiation – return to their original specialized form.
Transdifferentiation – converting one sort of cell into another;
capability of particular cells of the organ or tissue, including stem
and progenitor cells to differentiate into a different type of cell
(specific for different tissues or organs).
STEM CELLS
 totipotent (from the Latin totus = entire)
having unlimited capability – total potential
ability of a single cell to divide and produce all the differentiated
cells in an organism including totipotent cells
 pluripotent (from the Latin plures = many)
potential to differentiate into any of the three germ layers:
endoderm, mesoderm, or ectoderm
 multipotent
potential to give rise to cells from multiple, but a limited number
of lineages (related type, e.g. blood cells)
 unipotent
 progenitor – precursor
partly differentiated cells that divide and give rise to
differentiated cells
Differentiation
of human
tissues
Progenitor
and
stem
cells
STEM CELL
PROPERTIES
 undifferentiated cells (unspecialized)
none tissue-specific structures enabling specialization
 long-term renewal by cell division – proliferation
long-term self-renewal (unspecialized cells)
 under specific physiological or experimental
(laboratory)
conditions conversion into specialized cells – differentiation
external signals (genes)
internal signals (chemical from other cells,
physiological contact with other cells,
surrounding molecules)
STEM CELL
SOURCES

embryo (embryonic)
blastocyst

primordial germline cells in early fetal tissue
(embryonic germ cells)
gonadal ridge of a late embryo

foetus (fetal)
from terminated pregnancies

adult tissue (adult)
from organs, tissues
STEM CELL
TYPES
 embryonic stem cells – ES
pluripotent, (undifferentiated) cells from the embryo, potential to
become a wide variety of specialized cell types – give rise to all
derivatives of the three primary germ layers (mesoderm, ectoderm
and endoderm).
from early embryo (blastocyst); donated, in vitro fertilization or
therapeutic cloning (SCNT)
1981 – mouse stem cells
1998 – human stem cells (James Thomson, University of Visconsin-Madison)
 embryonic germ cells – EG
pluripotent cells from foetal tissue (gonadal ridge of a late
embryo), predicted for gonads development; also by humans
 foetal stem cells
from the foetus, terminated pregnancies, isolation from parts
predicted for particular organ development; more „directed“
STEM CELL
TYPES
 adult stem cells
undifferentiated (unspecialized) cell that occurs in a differentiated
(specialized) tissue
baby's cord blood
low number in baby's cord blood; clinical applications: blood cell
transplantation
bone marrow
EMBRYONIC STEM CELLS
Capability to give rise to
all derivatives of the three primary germ layers
(mesoderm, ectoderm and endoderm)
Welham MJ: Biochemist 28 (2006) 25-28
EMBRYONIC STEM CELLS
preparation
Embryo development
blastocyst
trophoblast – outer cell layer
blastocoel – cavity
inner cell mass
EMBRYONIC STEM CELLS
preparation
cell sampling

in vitro fertilization

embryo

blastocyst

izolation of inner cell mass (cca 30 cells)

stem cells

stem-cell culturing
subculturing and pasaging (cycles)

millions of cells
EMBRYONIC STEM CELLS
undifferentiated stage
LIF (Leukemia Inhibitory Factor)
responsible for the maintenance
pluripotency
STAT3 (transcription factor)
Oct4, Nanog – undifferentiated
EMBRYONIC STEM CELLS
differentiation
Embryoid Body
and subsequent spontaneous
differentiation
 chemical composition of media
(growth factors...)
 surface change
 gene introduction
EMBRYONIC STEM CELLS
properties
all criteria valid for mouse ES not for human ES









derived from the inner cell mass of the blastocyst.
long-term self-renewal.
pluripotent
diploid
capable of integrating into all tissues
transcription factor Oct-4 – non-differentiated
can be induced to continue proliferating or to differentiate.
lacks the G1 checkpoint in the cell cycle.
do not show X inactivation. (chromosomes)
EMBRYONIC STEM CELLS
characterization
Control requires
 undifferentiated
microscopically
 undifferentiated
surface markers, GFP
protein Oct-4 (transcription factor)
 chromosomes
microscopically (intact)
 proliferation after being de-frozen
 pluripotency
spontaneous differentiation
targeted (induced) differentiation into particular cell types
(tumor formation tested in repressed immunity mice)
ADULT STEM CELLS
Undifferentiated (unspecialized) cell
that occurs in a differentiated (specialized) tissue
Capable of
 self-renewal
 differentiation into specialized cells of tissues and
organs
ADULT STEM CELLS
multipotent
long-term self-renewal
malé množství
 bone marrow
hematopoietic stem cells (HSC)
mesenchymal stem cells (MSC)
endothelial progenitor (?)
 neural
 liver pancreas
 endothelial
 skeletal muscle
 epithelial
 brain
 blood
 blood vessels
 cornea
 dental pulp
 retina
ADULT STEM CELLS
normal differentiation
Differentiation into tissue
 hematopoietic  blood and immune system
erythrocyte, B and T lymphocytes, NK cells, neutrophils, basophils,
eosinophils, monocytes, macrofags, platelets
 mesenchymal  connective tissue, ligament
bone (osteocytes), cartilage (chondrocytes), adipose tissue
(adipocytes)
 neural in brain  neural tissue
neurons, astrocytes, oligodendrocytes
 epithelial  lining of the gastrointestinal tract
 skin
ADULT STEM CELLS
transdifferentiation (plasticity)
transdifferentiation (plasticity)
ability of stem cells from one adult tissue to
generate the differentiated types of another tissue
e.g.
 hematopoietic

 mesenchymal

 neural in brain

brain, skeletal muscle,
heart muscle, liver
chondrogenic, osteogenic,
myocytes, cardiomyocytes,
adipocytes, tendon
blood, skeletal muscle
ADULT STEM CELLS
transdifferentiation (plasticity)
generation of tissue type arising from the same or different
germ layer
same: bone marrow (mesoderm)  skeletal muscle (mesoderm)
different: bone marrow (mesoderm)  neuronal (ectoderm)
HEMATOPOETIC STEM CELL
MESENCHYMAL STEM CELLS
MESENCHYMAL STEM CELLS
differentiation
 depends on many factors
e.g. cytokines and other regulatory molecules concentration,
cell culture density and plating property-based selection,
mechanical forces application
 in vitro directed differentiation (promoted by application of
active substances)
Concentrations and conditions may differ for particular organism.
 different cell lineages including
osteoblasts, chondrocytes, adipocytes, fibroblasts, myoblasts,
cardiomyocytes, hepatocytes, neurons
MESENCHYMAL STEM CELLS
MESENCHYMAL STEM CELLS
applications
 tissue engineering
application directly into the defect or into
circulation blood
infused to the peripheral circulation have the
ability to migrate to a specific site of injury and
differentiated into the particular cell type
 gene therapy - genetic engineering
gene transducticion using viral vectors for
therapeutical altering abnormal gene function
MESENCHYMAL STEM CELLS
applications
 some applications …
defects of bone, cartilage, invertebral disc, heart tissue
regeneration,
nerves,
hematopoiesis
renewal,
suppression of immune rejection after implantation,
potential for kidney, muscle, skin regeneration
 ... and risks
clinical applications – MSCs fusion with endogenous
differentiated cells and formed tetraploid cells in vivo,
permit tumor growth in allogenic recipients; extremely
rare