Theoretical Grounds of Clinical Medicine
Significance and Perspectives of Stem Cells in Clinical Medicine II
Aleš Hampl
& Dáša Bohačiaková & Tomáš Bárta & Josef Jaroš
March 2018
Why people fell in love with stem cells?
(Embryonic, Adult, Induced)
Promise for
biomedicine
•Replacement therapy
•Drug development
•Disease modeling
•Toxicity testing
Food for thought
•Mechanism(s) of self-renewal ?
•Mechanism(s) of differentiation
•Symmetric/asymmetric division ?
•Pluripotency ?
?
?
Mother nature and scientists supply us with many
Stem cells generate and regenerate our body
1. Undifferentiated growth
2. Differentiation
Capability to differentite
into specialized
cell types
Pluri/Multipotency
Capability to produce
identical copies of itself
Self-renewal
Different properties
Fetal
Organ
Tissue
Embryonic
stem cells
Adult stem
cells
Induced pluripotent
stem cells
Cancer stem
cells
1998
2006
Fulfilling dreams needs solid grounds
Solid numbers
of cells
Safety
Reaching proper
cell phenotypes
3D organization
and integration
Scaffolds
?
Bioreactors
What will we discuss today ?
One have to be cautious - representative example of risk associated with propagation of stem cells outside the body
Stem cells in real clinic - example of what stem cells and how they are successfully used in tissue reconstruction
Lungs made from stem cells - two ways to go
Strong nerves made from stem cells - where we stand - example of the story, to which we also contributed
Twisting biology for good – new scenarios for driving stem cells to where we need them
Also stem cells need support and help - how to provide stem cells with the right and caring environment
What will we discuss today ?
One have to be cautious - representative example of risk associated with propagation of stem cells outside the body
Stem cells in real clinic - example of what stem cells and how they are successfully used in tissue reconstruction
Lungs made from stem cells - two ways to go
Strong nerves coming from stem cells - where we stand - example of the story, to which we also contributed
Twisting biology for good – new scenarios for driving stem cells to where we need them
Also stem cells need support and help - how to provide stem cells with the right and caring environment
Genetic changes develop in self-renewing hESC
2007
2010
October 2011
…. and also in adult stem cells
INTERPHASE METAPHASE ANAPHASE
NORMAL
ABNORMAL
Cultured hESC display centrosomal overamplification
that produces abberant mitoses
DAPI (chromatin)
pericentrine (centrosome)
a-tubulin (microtubules)
Holubcova et al., Stem Cells, 2010
Chromosome missegregation Chr. gain + loss
G1
G2
SM
c
d
f e
a b
g
Why this may represent a serious problem ?
.... with very high frequency !
Undifferentiated hESCs
Brno
Stockholm
Boston
Holubcova et al., Stem Cells, 2010
cell
line
passage
number
mitoses
multicentrosomal / total
multicentrosomal mitoses
percentage
B10/CBA_11.1 P8 5 / 120 4,17 %
B10/CBA_11.2 P5 3 / 122 2,45 %
B10/CBA_11.3 P8 3 / 96 3,13 %
B10/CBA_11.4 P5 0 / 104 0,00 %
B10/CBA_11.5 P7 1 / 111 0,90 %
B10/CBA_11.6 P7 0 / 47 0,00 %
B10/CBA_11.7 P3 1 / 125 0,80 %
B10/CBA_11.8 P4 3 / 109 2,75 %
cell
line
passage
number
mitoses
multicentrosomal / total
multicentrosomal mitoses
percentage
B10/CBA_11.1 P8 5 / 120 4,17 %
B10/CBA_11.2 P5 3 / 122 2,45 %
B10/CBA_11.3 P8 3 / 96 3,13 %
B10/CBA_11.4 P5 0 / 104 0,00 %
B10/CBA_11.5 P7 1 / 111 0,90 %
B10/CBA_11.6 P7 0 / 47 0,00 %
B10/CBA_11.7 P3 1 / 125 0,80 %
B10/CBA_11.8 P4 3 / 109 2,75 %
In mESC the frequency of multicentrosomal
mitoses is low
unpublished
Somatic cells hiPSC
fibroblast source
multicentrosomal
/ total mitoses
multicentrosomal
mitoses
percentage
clone ID (passage
number)
multicentrosomal /
total mitoses
multicentrosomal
mitoses
percentage
Human foreskin
fibroblasts
0/96 0,0% HFF_L1 (P20) 10 / 110 9,09%
HFF_L2 (P20) 5 / 125 4,0%
Normal human
dermal
fibroblasts
(Lonza)
6/60 10,0% NHDF (P26+7) 14 / 202 6,9%
Adult dermal
human
fibroblasts
2 / 267 0,74% AHDF_#1 (P36 25/249 10,07%
AHDF_#4 (P35) 29/217 13,36%
Ligase IV
mutated
(patient derived)
0 / 60 0,0% FO7/614 (P18+10) 5 / 110 4,5%
4 / 111 3,6%
FO7/614_shRNAp53
(P20+11)
29 / 174 16,6%
0 / 52 0,0% GM16088 (P19+9) 1 / 77 1,29%
0 / 56 0,0% GM17523 (P18+6) 20 / 160 12,5%
In hiPSC the frequency of multicentrosomal
mitoses is variable depending on cell line
unpublished
undiff. hESC
DAPIOct3/4mergetransmitted
3D RA 5D EB + 10D
A
C
0
5
10
15
hESC 5D EB
+ 10D
%ofmulticentrosomalmitoses
0
5
10
15
%ofmulticentrosomalmitoses
hESC 3D RA
B
Differentiated cells
Supernumerary
centrosomes develop
only in pristine hESCs
Holubcova et al., Stem Cells, 2010
0
5
10
15
20
25
30
0 50 100 150 200 250 300 350 400
Multicentrosomalmitoses(%)
Passage number
Prolonged culture reduces the frequency of
mitoses with supernumerary centrosomes
Holubcova et al., Stem Cells, 2010
Supernumerary centrosomes have normal structure
bipolar multipolar
pericentrin
a-tubulin
DNA
Figure S3
Holubcova et al., Stem Cells, 2010
4C 8C >8C
Both endoreduplication and mitotic failure contribute
to overamplification of centrosomes in hESC
0%
20%
40%
60%
80%
100%
>8C
8C
4C-8C
4C
Low High
CCTL10 CCTL12
Low High
CCTL14
Low High
kinetochores
Holubcova et al., Stem Cells, 2010
34 hours
20 hours
20 hours
14.5 hours
16 hours
unpublished
Cells divide unfaithfuly
What will we discuss today ?
One have to be cautious - representative example of risk associated with propagation of stem cells outside the body
Stem cells in real clinic - example of what stem cells and how they are successfully used in tissue reconstruction
Lungs made from stem cells - two ways to go
Strong nerves coming from stem cells - where we stand - example of the story, to which we also contributed
Twisting biology for good – new scenarios for driving stem cells to where we need them
Also stem cells need support and help - how to provide stem cells with the right and caring environment
left breast ablated
because of cancer
7 months after lipofilling
2x consecutive lipofilling
(lipomodelation)
Department of Plastic and Cosmetic Surgery
(Dr. Streit)
Missing connective tissues can be supplied by
grafting adipose tissue
Missing connective tissues can be supplied by
grafting adipose tissue
What is the concept ?
fat grafting = a promising surgical technique that uses patient’s own fat
for tissue regeneration and augmentation
What is behind the effect ?
adipose-derived stem cells (ASCs) = potent fat tissue cells responsible for regeneration
What is the question ?
various ways of fat processing = do they produce the same cells ?
= do they give the same clinical outcome ?
Teaming up for getting the answer
Department of Plastic and Cosmetic Surgery
&
Department of Histology and Embryology
Patients & Collection of adipose tissue
Liposuction abdominoplasty
Application
500 ml normal saline
+
epinephrine
Liposuction
150 ml of lipoaspirate
(via 3.5 mm cannula)
Transfer to the lab
average length of
ischemia 2.5 hr at 37°C
Processing of lipoaspirate
Sedimentation
• separation by gravity
• still used
• control group
• time consuming
• upper fat layer
• Fat fraction 71%
Centrifugation
(1200 g, 3 min)
• Upper oily layer
• Fat layer - 56%
Low density (upper 2/3)
High density (bottom 1/3)
• Liquid layer
• Pellet
Membrane-based
tissue filtration
(PureGraftTM membrane)
• 2x washing 2x30 ml PBS
• filtration 2x 5 min
• Fat fraction - 46%
Many different characteristics were determined
Viability of cells
sedimented membrane centrifuged centrifuged pellet
filtered low dens. high dens.
NO difference
Microstructure of individual preparations
cell debris
lipid droplets
adipocytes + amorphous component of ECM
adipocytes
fibrilar component of ECM
erythrocytes
Significant differences
Some features
Sedimentation:
• abundant debris + oil drops
Centrifugation:
• minimum debris + oil drops
• reduced amount of ECM
Filtration:
• minimum debris + oil drops
• minimum ECM
Pellet
• abundant ECM + erythrocytes
native AT pellethigh dens.low dens.filteredsedimentedlipoaspirate
Molecular phenotype of adipose-derived stem cells
Perfectly normal in all preparations
Negative for:
CD31 & CD45
Positive for:
CD90 & CD105
CD 90 + CD 105 +
Proportion of adherent adipose-derived stem cells
SOME differences
calculated as number of SC in 1 ml of original processed adipose fraction
sedimented membrane centrifuged centrifuged centrifuged pellet
filtered low dens. high dens.
*
Capacity to differentiate towards
adipogenic and osteogenic
Perfectly normal differentiation capacity in all preparations
non-differentiated control differentiated
adipogenic
(oil red)
osteogenic
(alizarin red)
Key medically relevant findings
Sedimentation
• very gentle – lowest yield of ASC
• time consuming – unpractical for clinical setting
Viability of cells
• independent of the procedure
• probably much more affected by the duration of ischemia (manipulation)
Centrifugation x Membrane-based filtration
• quantitatively and qualitatively very similar outcome
Centrifugation seems to be preferable
uncompromised quality & cost effectiveness
It works and a spectrum of applications is wide
persistent postradiation
ulceration (sarcoma treament)
2x application of the graft
(arround + underneath the ulcer)
7 months after grafting
Unpublished (Dr. Streit)
What will we discuss today ?
One have to be cautious - representative example of risk associated with propagation of stem cells outside the body
Stem cells in real clinic - example of what stem cells and how they are successfully used in tissue reconstruction
Lungs made from stem cell - two ways to go
Strong nerves coming from stem cells - where we stand - example of the story, to which we also contributed
Twisting biology for good – new scenarios for driving stem cells to where we need them
Also stem cells need support and help - how to provide stem cells with the right and caring environment
Stem cells can repair adult tisues/organs
Reparative behavior
- Constitutive high rate
- Defined hierarchy of
stem/progenitor cells
Epidermis
Intestine
Blood
- Low steady-state turnover
- Robust repair after damage
Lung
Liver
Pancreas
- Inefficient
- Scaring instead of repair
Brain
Heart
Ciliated cells
Goblet cells
Serous cells
Neuroendocrine cells
Basal cells
Club cells
Ciliated cells
Basal cells
Pneumocytes type II
Pneumocytes type I
Lungs as one of the targets
More than 40 cell lineages identified in lungs !!!
Anterior ventral foregut endoderm + Mesoderm
Vascular smoth muscle
Airway smooth muscle
Cartilage
Fibroblasts
Pericytesproximal
distal
Lung diseases potentially treatable by cell therapies.
Lung disease Affected components Therapeutic target
Respiratory distress syndrome
Alveolar epithelium
Capillary endothelium
Epithelia and endothelia
regeneration
Asthma
Epithelium
Myofibroblast
Airway smooth muscle
Inhibition of inflamation
Inhibition of airway remodeling,
Inhibition of muscle heperplasia
Bronchopulmonary
dysplasia
Alveolar epithelium
Capillary endothelium
Interstitial fibroblasts
Inhbition of inflamation
Regeneration of alveolar septa
and epithelium
Cystic fibrosis Airway epithelium
Delivery of CFTR
(cystic fibrosis conductance regulator)
Chronic obstructive pulmonary
diseases (emphysema)
Alveolar epithelium
Capillary endothelium
Interstitial fibroblasts
Generate 3D alveolar structure
Bronchiolitis obliterans
Airway epithelium Regeneration of epithelia
Cancer All components
Complete replacement of 3D
structure
and others
Respiratory diseases are the third leading cause of death in the industrialized world.
Lung replacement is often the only solution.
Options for new therapeutic strategies
Acute alveolar damage
• inhalation injury
• blast injury
Activation of healing
potential of resident
progenitors
Chronic lung damage
• chronic obstructive pulm. disease
• fibrosis
• bronchopulmonary displasia
• and others
Cellular therapy Lung engineering
What cell sources we may consider ?
2
Stem / progenitor cells
residing in lung tissues
1
Adult stem cells isolated
from non-lung compartmets
3
Lung stem / progenitor
cells differentiated from
pluripotent stem cells
Anas Rabatta
Stem / progenitor cells residing in lung tissues
Dr. Zuzana Koledová
BASCs
variant Clara cells
Clara cells
Bronchiolar-Alveolar SC
Collect lung
Chop lung
Enzymatic
digestion
Differential
centrifugation
Lung epithelial organoids
Mesenchymal cells
Single-celled
lungepithelialcells
Culture in
non-adherent conditions
Enzymatic
digestion
Primary spheres
Secondary spheres
Enzymatic
digestion
Culture in
3D Matrigel / collagen Culture in 3D
Matrigel
Isolation + Characterisation of mouse LSPC
Efficiency of Primary
lungospheres
formation
The number of
(EpCAM+,CD49f+,
CD104+,CD24low) cells
Viability
The
number
of cells
before
sorting
2.1%183486.8%35*106
Liberase
Lungospheres originating from one single cell
unpublished
Morphogenesis in lungospheres grown for
2 weeks in suspension culture
unpublished
E-cadherin DAPI cytokeratin-14 DAPI Pro-SP-B DAPI
Direct differentiation of pluripotent SC
into airway epithelia
+ Activin
+ FBS
- Activin
- FBS
+ FBS
+ FGF2
+ KGF
+ EGF
D0 D6 D15 D28
Endoderm induction Anterior-posterior
patterning
Tissue specification
Vitronectin
hESC
3D Organotypic cultureAir-Liquid interface culture
unpublished
Direct differentiation of pluripotent SC
into airway epithelia – 15 days
Light H+E Sox17 TTF1
Anterior ventral foregut endoderm TF
Air-Liquid interface culture
SPA
pro-SPB
0 4 6 8 10 12 14 21 23 26 28
H441
Pneumocytes II
Days
Pro-SPBSPACCSP
Pneumocytes IIClub cells
unpublished
Direct differentiation of pluripotent SC
into airway epithelia – 20 days
Microvili Cavities
Air-Liquid interface culture
unpublished
Direct differentiation of pluripotent SC
into airway epithelia – 20 days
Polarized cells
Tight junctions
Microvili
Flattened cells
Lamellar bodies
Air-Liquid interface culture
Alveolar-like organization
0,5 – 3 mm
unpublished
Direct differentiation of pluripotent SC
into airway epithelia – 20 days
3D Organotypic culture
Analysis in progress
Organoids
unpublished
Differentiation of early lung progenitors (ELP) from hESC
FBS 10 % 10 % 0 2 %
Activin A - + - ITS
- - + +
FGF2 - - - +
EGF - - - +
Heparin - - - +
Vitronectine + + + +
0 6 151Day
Definitive
endoderm
Anterior
ventral
foregut
endoderm
hESC
unpublished
Hana Kotasová
Early lung progenitors X Common endoderm progenitors
Lamellar bodies
Foxj1 Ciliated cells
p0 p20 p30 D7 D15 D21 H441
Self-renewal Differentiation
Organoid (10 days 3D)
AQP5CCSPSPA
Differentiation for 20 days (ELP in p20)
Pneumocytes II Pneumocytes IClub cells
unpublished
Early lung progenitors differentiated for 25 days in matrigel
unpublished
H+E
Simple squamous epithelium
(alveolar-like)
Pseudostratified epithelium in tubular structures (airway-like)
AQP5
Pneumocytes I
pro-SPC CCSP
Club cells
Pneumocytes II
Club cells
Col IV
Phalloidin
DAPI
+ respond to topography by adjusting their shape
unpublished
Early lung progenitors attach and proliferate on mouse
decellularized lung scaffold
unpublished
zoom
ELP differentiate when transplanted under kidney capsule
ESC can give rise to highly organized
organ-like structures
April 2011
3D culture (EB)
+ integrins
+ laminin
+ entactin
+ Nodal
Internal nuclear layer
External nuclear layer
Ganglion cells
D24
What will we discuss today ?
One have to be cautious - representative example of risk associated with propagation of stem cells outside the body
Stem cells in real clinic - example of what stem cells and how they are successfully used in tissue reconstruction
Lungs made from stem cells - two ways to go
Strong nerves coming from stem cells - where we stand - example of the story, to which we also contributed
Twisting biology for good – new scenarios for driving stem cells to where we need them
Also stem cells need support and help - how to provide stem cells with the right and caring environment
Two sorts of diseases that disable central nervous system
Acute:
• spinal cord injury
• brain trauma
• stroke…
Chronic:
• Parkinson’s Disease
• Alzheimer’s Disease
• Amyotropic lateral sclerosis
Any posssible help from stem cells ?
Model the disease
+ discover new drugs
Trophic support for the
surviving / damaged cells
Replace lost +
non-functional cells
All kinds of cells can be considered:
• embryonic + induced pluripotent stem cells
• fetal neural cells - problematic
• adult neural stem cells - problematic
Neural differentiation of pluripotent SC – recapitulating development
Carlson, Human Embryology and Developmental Biology, 2014
neural rosettes
Grabiec et al. 2016
What else you can do?
neural rosettes
2D
3D
neural stem cells
(selfrenewing)
mature neurons/glia
spheroids cerebral organoids
(whole brains)
Nature, 2013
Many diffrent ways of getting there
… growth factors:
•Peripheral neurons
Coculture with mouse
stromal cell line PA6
•GABAergic neuronsNeurotrofin-4
•Dopaminergic neurons
FGF8/Shh, ascorbic acid
+ BDNF
•Motoric neuronsFGF2, RA, Shh
•Astrocytes
CNTF (ciliary neurotrophic
factor)
•OligodendrocytesCNTF/PDGF
… culture surface/scaffolds:
collagen, laminin,
fibronectin
integrins, NCAM
elasticity / plasticity
2D / 3D
Nisbet et al., 2008
… by combination
of these
Problem of
heterogenous
differentiation
Can we convert this knowledge into medical benefit ?
Hans Keirstead
UC Irvine
2005 2010 2015
Data published in
Journal of
Neuroscience
1st Clinical Trial
with hESCs
Approved
Clinical Trial
Cancelled
Clinical Trial
Re-Approved
2014
1st positive
efficacy data
form 4 patients
Sept. 2016
1990
Hans Keirstead
started his PhD in
Neurobiology
11 years
26 years
Spinal cord injury
• spinal cord trauma destroys numerous cell types
• in most cases of injuries, spinal cord is not damaged completely and some
neuronal connections remain intact
• oligodendrocytes (new myelinisation) may improve the function of motoneurons
Further study towards cell therapies for acute CNS injuries
The goal:
To obtain clinically applicable Neural Stem Cell line from hESC
Department of Anesthesiology and Stem Cell Program,
University of California San Diego, La Jolla, CA
Martin Marsala, MDDasa Bohaciakova
(Dolezalova), PhD
1) Derivation method is simple, robust, cost-effective and can be implemented in
cGMP
• no xenobiotic tissue culture components or enrichment methods
2) Cell line of NSCs is expandable (and karyotypically stable), well characterized in
vitro, homogenous, and has broad differentiation potential
• Methods generate heterogeneous population of NSCs enriched with “contaminating” cells with limited
differentiation and/or proliferative capacity
3) Extensive in vivo studies
• Several reports suggest that tumorigenicity or neural overgrowth in vivo may represent a major obstacle in
the future application of hESC-derived NSCs into human therapies
Key requirements on clinically applicable Neural Stem Cells
• After transplantation in vivo, cells survive and integrate into host tissue and
differentiate into mature neurons and/or glia and do not form any tumors
• Method has successfully been transferred to cGMP facility at UC Davis
• Several NSC cell lines are currently being tested on Amyotrophic Lateral Sclerosis
(ALS) and Spinal cord injury animal models
Shortcut to the current stage of the story
Injection site
Some more neurobilogy fun here in Brno
Martin Barak
P-POOL
Dasa Bohaciakova
(Dolezalova), PhD
to develop robust protocol for differentiation
and culture of 3D cerebral organoids from
hiPSC for modeling Alzheimer’s disease
Modeling Alzheimer’s disease in the dish
Where we are now ?
Organoids with visible mature structures
What will we discuss today ?
One have to be cautious - representative example of risk associated with propagation of stem cells outside the body
Stem cells in real clinic - example of what stem cells and how they are successfully used in tissue reconstruction
Lungs made from stem cells - two ways to go
Strong nerves coming from stem cells - where we stand - example of the story, to which we also contributed
Twisting biology for good – new scenarios for driving stem cells to where we need them
Also stem cells need support and help - how to provide stem cells with the right and caring environment
Twisting biology for good
new scenarios for driving stem cells to where we need them
Tomáš Bárta
Differentiation potential
Differentiation potential
Ectoderm
Mesoderm
Endoderm
Pluripotent
Multipotent
typeA
typeB
typeC
typeD
How to change the cell fate?
Differentiated
Cell reprogramming into pluripotent state
Oct4, Klf4, Sox2, c-myc
Somatic cells (fibroblast) Induced pluripotent stem cells
Extrinsic conditions
(e.g. growth factors,
cell culture conditions etc.)
Advantages: patient-specific cells
Disadvantages: instability of the genome, tumorigenicity, high-costs, safety
Sir John B. Gurdon – 1958 - 1966
Ian Wilmut - 1997
“This result is of interest since it shows
that genetic factors required for the
formation of a fertile adult frog are not
lost in the course of cell
differentiation...”
Recipient cell contains factors that are
capable to revert somatic cell into
embryonic cell.
X
Sir John B. Gurdon – 1958 - 1966
“We describe here some adult frogs
which are derived from transplanted
intestine nuclei and some of which are
fertile.”
Oct4
Sox2
Klf4
c-Myc
24 genes, specific for
pluripotent stem cells
he always took out one gene and followed the efficiency of
reprogramming => 10 genes seriously affected the efficiency.
10 genes, specific for
pluripotent stem cells
he selected 4 genes
Shinya Yamanaka - 2006
Shinya Yamanaka – 2006 - 2007
Sir John B. Gurdon - 1958
Ian Wilmut - 1997
?
How only 4 genes can reprogram the fate of a cell?
What is happening during the reprogramming?
D0 D3 D6 D9
D12 D15 D18 D21
I. Shut down of genes maintaining the “identity” of fibroblasts.
I. dedifferentiation and upregulation of genes maintaining proliferation
II. MET – transition from mesenchymal to epithelial phenotype.
III. Establishment of pluripotency gene network.
Increasing the safety of the cell reprogramming
Graf, 2011
Conversion of one cell type to another cell type.
Easily accessible and easy-to-cultivate cell types (fibroblasts, blood cells) are often used.
Transdifferentiation
Advantages: patient-specific cells
Disadvantages: low-efficiency, restricted proliferative capacity, limited cell type diversity, senescence,
and do not generally produce progenitor cells
adenovirus encoding PDX-1
injected to liver
diabetic mice
Transdifferentiation of hepatocytes into insulin producing cells.
PDX-1 Insulin
Activation of insulin expression in human hepatocytes infected
with virus encoding PDX-1
Pdx-1 (green) a insulin (red)
Activation of insulin promoter (green)
Ascl1, Brn2, Myt1
actinαMHC
Gata4, Mef2c, Tbx5
D0 D6
Bmp4
D18 D24
FGF and GSK3 inhibitionFGF and GSK3 inhibition
Culture
medium
without FGF
Culture
medium
without FGF
10% FCS,
Retinoic acid,
Taurine
10% FCS,
Retinoic acid,
Taurine
Spheroids generation Induction of neural retinal epithelia Retinal pigmented epithelium formation Photoreceptor maturation
Ncad MITF Pax6 Opsin CRX
Oct4
Sox2
Pax6
Rax
MITF
β-actin
D0 D7 D14 D21 D28 D35 D42 D49 D56
What we are trying to achieve?
Clinical applications
? ?
? ?
?
?
?
?
?
?
?
?
Many issues must be addressed....
Basic research
?
Take-home message
typeA
typeB
typeC
typeD
There are many ways...
...we are trying to find the safest and cheapest (the best?) way.
What will we discuss today ?
One have to be cautious - representative example of risk associated with propagation of stem cells outside the body
Stem cells in real clinic - example of what stem cells and how they are successfully used in tissue reconstruction
Lungs made from stem cells - two ways to go
Strong nerves coming from stem cells - where we stand - example of the story, to which we also contributed
Twisting biology for good – new scenarios for driving stem cells to where we need them
Also stem cells need support and help - how to provide stem cells with the right and caring environment
Caring MicroEnvironment
how to provide stem cells with the right and caring environment
Josef Jaros
For what it is applicable in medicine?
Cancer biology
Stem cell biology
Disease modelling
Developmental biology
Molecular processes
Drug testing
In vitro
cultivation
Cells
Matrix
Enviro
Soluble
(growth)
factors
Factors of environment
Comparison of environments
Pros & Cons
+ Manipulation & analysis
- Artificial conditions
- Cells flat
- Nutrients &soluble factors from 1 side
- Connected to other cells 5-15%
- etc.
Diverse cells behavior and reactivity to drugs grown in
2D and 3D environment
bone cartilage
fat skin intestine
nerves heart muscles
Different tissue
Different structure
Adhesion – Proteins, peptides
Topography – fiber diameters
Mechanosensing – mesh size, stiffness
Cell Motility – Porosity
Molecule Diffusion - mesh size
Matrix Degradability - MMPs
Solution
- Self-organization – e.g. organoids, spheroids
- Surface modifications of Petri dish
- Building our own 3D matrix
Cells need 3D ECM
In vitro In vivo
Self-organizing cells
Organoids & Spheroids formation
Differentiation in 3D = Organoids in culture
- Intestinal, cerebral, lung…
Clevers, Knoblich, …
3D Electron microscopy visualization of
spheroids (Jaros et al, 2017)
Non-adherent surface
V, U shaped well
Surface modification and 3D matrix
Natural
• Natural recognition by cells
• Expensive isolation
• Availability and purity
– Individual proteins ECM (collagen, fibronectin, etc.)
– Mixture – Matrigel, Geltrex, …
– Decellularized tissue (Preserved structure)
Heart
Synthetic
• Precisely defined
• Can be customized
• Large scale production -> low price
• Easy modifications
– Polymers (PLLA, PCL, PU, …)
– Peptides
– Foams, mesh fibers, scaffolds, hydrogels
Fibronectin mesh
Ott, H.C. et al, Nat Med, 2008
Decellularized tissue - mouse lungs
Collagen
Fibronectin
Laminin
DecellularizedComplete
Immunostained ECM
Blue – cell nuclei
Hematoxylin
Eosin
Alv
Alv
Alv
Basal
lamina
Airway
Collagen
Elastin
+
Fibrilin
Zuzana Garlíková
Hana Kotasova
Decellularized lungs
preserved anatomical structure
DecellularizedComplete
Alveolar
region
Vessel
Vessel
Bronchiolus
Airway
Garlikova, Hampl et.al. , Tissue Eng., 2018
Man-made structures
Cultivation medium
Viscosity play role for cells
Hydrogels
formed by crosslinking or polymerization
Hyalyronan - Plaster, patch
Patterson J. et al, MaterialsToday, 2010
Fibrilar
structure
collagen I Fibrin
matrix
Hydrogel of
modified
hyaluronic
acid
Synthetic
peptide
Hydrogel
RADA
Hematology - MethoCult – growing of colonies
Effort to produce materials
with structure close to natural ECM
Injectable,
>98% of water
content
Nanofibers
• Soluble materials, spun fibers under 1 um
• Mostly for covering – skin, endothelium, dura mater…
Sedlakova, submitted, 2017
Modified PCL and PLLA nanofibers
Porous scaffolds
Structure
● Pores (60-300 um)
● Diffusion
● Degradability
● Dynamics – release of chemicals –drugs,
molecules for differentiation (growth
factors, morphogens)
250 um
Application in clinics
• Trachea
• Heart valves
• Bones
Jaros, unpublished
3D cultivation troubles
• Manipulation, analysis, sterilization
• How to get cells inside
• How to get cells/proteins out (PCR, WB, etc.)
• How to provide nutrient/waste exchange
• Organization of cells
Golunová A., Jaros J., et al., Biomaterials, 2015
Proks, V., Jaros, J., et al., Macromol Biosci 2012
3D porous
synthetic gel
250 um
3D bioprintingsolution
3D bioprinting allows cell organization and
tissue formation
- Extruders and ink-jetting
- Hydrogels applied
- Human stem cells and progenitors
- Combination of material and cells
Organization and positioning of cells is important aspect
for controlling cell behavior
Josef Jaros
Karolina Spustova
Richard Mackovic
Printed spheroids
Picoliter drops arrays
10 mm
Hydrogel vessels
tubes with walls of 300 um
Jaros, unpublished
Branching structure
Jaros, unpublished
Printing cells
into shapes of branched tubes
Jaros, unpublished
Take-home message
Caring microenvironment and organization of stem cells help to
build tissue structures and to understand biological mechanisms..
Video
Dynamics of growth of hESC spheroids
organized within hydrogels
by 3D bioprinting