Theretical Fundamentals of
Clinical Medicine
Significance and Perspectives of the Stem Cells in Clinical
Medicine I
Vladimír Rotrekl 2017
Stem Cell (SC): criteria and definition
Selfrenewal
Diferentiation
Ability to create its
copies
Ability to change its
properties and
differentiate
Clonal capacity
•Symetrical division
•Asymetrical division
Toti
Pluri
Multi potent
Oligo
Uni
Stem cells self-renew, proliferate
Self-renewal = the most important property of stem cells; ability to
produce identical copies
Symetrical devision Asymetrical devision
Combination of both mechanisms = neural SC
Cell death
Embryonic SC Fetal and adult SC (exceptions exist)
.... SC differentiate and regenerate tissues
totipotent pluripotent multipotent oligopotent unipotent
zygote Embryonic SC Hematopoetic SC Gastrointestinal SC Prostate SC
CFU-GM
CFU-E & BFU-E
CD34

Neurons
Non differetiated SC
Just examples
Colony of thousends of cells
Primitive ectoderm/epiblast Trofectoderm
Polar trofectoderm
Extraembryonal ectoderm
Chorionic ectoderm
Trofoblast placenty
Mural trofectoderm
Ectoplacentálnal cone
Giant cells of trophoblast
placenta
Parietal
yolk sac
Primitive entoderm
Ectoderm
• neural tissue– neural SC
• skin – skin SC
Mesoderm
• bone marrow and blood– hematopoetic and mesenchymal SC
• muscules and bones – tissue specificSC
Entoderm
•lungs, liver, pancreas – organ/tissue specific SC
• larynx, stomache, intestine – intestinal SC
Visceral entoderm
Parietal entoderm
Embryonic SC
Origin and developmental ontogenesis of stem cells (SC)
Primordial stem cells
• gametes
Stem Cells in Medicine
• Pluripotent
embryonic and induced…
• Mesenchymal (Dr. Pešl)
clinical trials and stem cell turism
• Hematopoeitic (prof. Krejčí)
mostly hematooncologic and imune
disorders
Stem Cells in Medicine
• Pluripotent
embryonic and induced…
• Mesenchymal (Dr. Pešl)
clinical trials and stem cell turism
• Hematopoeitic (prof. Krejčí)
mostly hematooncologic and imune
disorders
Pluripotent stem cells
One Ring to Rule Them All…
…or shall it be:
One cell to rise them all…?
What do we mean by EMBRYO, when
talking Stem Cells
• preimplantation stage
• blastocyst 4 days old
• composed of several
dozens of cells
• inner cell mas
~9000 embryos implanted (typicaly 3 at the time) anualy in CR
rest >50% remains frozen …
"[T]he bottom line is that there are 400,000 frozen
embryos in the United States, and a large
percentage of those are going to be thrown out.
Regardless of what you think the moral status of
those embryos is, it makes sense to me that it's a
better moral decision to use them to help people
than just to throw them out. It's a very complex
issue, but to me it boils down to that one thing."
James Thompson
The destruction of human embryos to harvest stem
cells is "not only devoid of the light of God but is
also devoid of humanity" and "does not truly serve
humanity."
Benedict XIV
Act 227/2006 Sb
Act on the human embryonic stem cell research and related issues
- Governmental registry of the hESC lines
- Research only with the aproval of Ministry of Youth and Schools
- Research must not lead to human being creation (cloning)
Act 273/2011 Sb
- Embryo storage for infinite time (EU usually 5 yrs)
- Embryos discarded after 10 yrs, if the pair does not wish othervise
Act 227/2006 Sb
Possible CATCH 22…?
„Frozen generation“ – circa ½ million frozen embryos in US
CR: embryo storage (ČR) …. ~ 10 000 CZK (single payment - usually)
in 2007 – 3400 IVFs
§9 par 1: a) extra embryo may be used for research only with written consent from
both parents signed before embryonic stem cell derivation.
Act on the human embryonic stem cell research and related issues
Blok I - discussion
• Is this topic relevant to med students?
• Are the arguments about human
existence from conception till birth
relevant and are the arguments about
the absence of certain abilities of the
embryo relevant (e.g. Ability to think ad
feel pain/stress)?
• (in)sufficient legislature concerning
hESC in CR?
Albert Lasker basic medical
research award 2009
Induced Pluripotent Stem Cells IPSC
(Yamanaka 2006)
Thy1 (and other genes typical for fibroblasts))
0 4 8 12
retroviral activity
Pluripotency markers
Pluripotency genes
Telomerase activity
Inactivation of X chromosome
Somatic cell
i.e. skin fibroblast
iPS cell
Time in days
Stable reprogramming to stemness
Alternative source of pluripotency - iPS cells
- SC created from somatic (i.e. differentiated) cells using genetic manipulation
Kinetics of fibroblast reprogramming into the pluripotentnt SC
Oct4
Sox2
c-Myc
Klf4
iPS are indeed capable of chimera formation
Oct-4 GFP
fibroblasts
Oct-4 GFP
iPS
Nanog GFP
iPS
Injection of chimera
(white and gray hair)
Nanog GFP iPS chimera Oct-4 GFP iPS chimera
Brambrink et al. Cell Stem Cell, February 2008
Induced pluripotent stem cells
Promissing in future medicine - patient-specific cells
Ethical problem with hiPSC?
• No need to destry human embryo
• Patient specific cells as the source for
reprogramming – limits the risc of GVH
Human Induced Pluripotent Stem Cells
Germ cells out of hiPSC
Diferentiation into PGC
Spermatocyte
Oocyte
Preimplantation embryo
Hubner, Science, 2003
?
Ethical problem with hiPSC …. I
• Altruistic sentiment and benefit expectation
versus
• Privacy protection
• Establishment of permanent (immortal) cell line (i.e.HeLa)
• Human cells and tissues comercionalisation
• Possibility to produce gametes/partenogenesis (germ cells)
• Genome editting before returning to the patients
Ethical problem with hiPSC …. II
US
UK
Interspecies chimeras for
human organ
transplantations
Blok II - discussion
• Is reprogramming relevant to med
students?
• Arguments about the human uniquness
in relation to cloning..
• (in)sufficient legislature concerning hiPS
in CR?
Rational design of the novel pluripotent stem cell
applications in clinics requires
• Opened and truthfull communication with public and stakeholders
• Buletproof patient consent protecting both the researchers as well as the patient
• Absolute transparency of the scientific results and preclinical tests
• Confidence in the systém and individuals (scientists and MDs)
What is this for?
Regenerative and
reconstructive medicine
Disease modelling
Understanding
pathological processes
Drug development
What is this for?
Regenerative and
reconstructive medicine
Disease modelling
Understanding
pathological processes
Drug development
Any PSC aplication requires quantitative differenciation into the target cell type
Relation between tumorogenicity and pluripotency
(a) telomerase
(b) Absence of contact inhibition
(c) Strong antiapoptotic aparatus
(d) High proliferation
Kooreman, J R Soc Interface. 2010
Current standard for clinical trials: Max 1 pluripotent cell per 106 differentiated cells
in the off-the-shelf product!
Primitive ectoderm/epiblast Trofectoderm
Polar trofectoderm
Extraembryonal ectoderm
Chorionic ectoderm
Trofoblast placenty
Mural trofectoderm
Ectoplacentálnal cone
Giant cells of trophoblast
placenta
Parietal
yolk sac
Primitive entoderm
Ectoderm
• neural tissue– neural SC
• skin – skin SC
Mesoderm
• bone marrow and blood– hematopoetic and mesenchymal SC
• muscules and bones – tissue specificSC
Entoderm
•lungs, liver, pancreas – organ/tissue specific SC
• larynx, stomache, intestine – intestinal SC
Visceral entoderm
Parietal entoderm
Embryonic SC
Origin and developmental ontogenesis of stem cells (SC)
Primordial stem cells
• gametes
Thomas Graf & Tariq Enver Nature 462, 587-594 (2009) doi:10.1038/nature08533
What determines differentiation in vivo and in vitro:
Cell line differentiation of embryonic stem cells in complex landscape of epigenetic
bariers (=cells are unstable on the hills), mountain platos (=RELATIVELY STABLE BUT
REVERSIBLE CELL STATUS) and deep walleys (=TERMINAL DIFFERENTIATION)
DIFFERENTIATION OF PSC – embryo development analogy…
Retinoic acid
AP axis establishment
Ribes, Development 2009 Zhang, PLOS 2015
RA ectoderm formation from PSC
DIFFERENTIATION OF PSC INTO FUNCTIONAL CARDIOMYOCYTES…
Pešl, Heart and Vessels, 2014
BMP4 helps to
polarize the
embryo during
gastrulation
(primitive
mesendoderm)
Hensen node with
FGF2 triggers
cardiogenesis
Activin A
triggers
mesoderm
formation
VEGF is
needed for later
embryo heart
morphogenesis
(ventricle
formation)
IWR inhibits Wnt
signal – prevents
neurodifferrenciat
ion etc.
Cardiomyocytes
starting spontaneous
beating
1mm
APD90(ms)
AP atrial ventricular
WT
DMDWT
DAPI / troponin T/C DAPI / α-actinin
DAPI / Troponin T/C /
α-actinin
FUNCTIONAL CHARACTERISATION OF PSC DERIVED CARDIOMYOCYTES…
Atomic Force MicrscopyCa2+ Transients
Cardiac Markers & Morphology Electrophysiology (Patch Clamp)
Department of Biology MU
Advances in hIPSC differentiation –
biological models and aplications
Neurons, astrocytes, oligodendrocytes
Cardiomyocytes
Insulin-producing pankreatic cells
Blood cells
Immunocompetent cells
Endotelial cells
Trofoblast cells
Respiratory cells
Osteoblasts
Hepatocytes
Melanocytes
Prostate cells
Germ cells
Example: thorasic spinal cord injury, myelopaty and SC treatment
Damage before the
treatment
After the treatment
Result: reconstruction of gray and white matter
reconstruction of motoric neurons
return of the mobility
Model: rat
Adapted from Stem Cells, 2010
Makrofág
Astrocyt
Oligodendrocyt
Axony s myelinem
Progenitory oligodendrocytů
Progenitory motoneuronů
…Heart patches…
Patient suffering
heart failure
5% ejection fraction
30% ejection fraction
… patient leaves the ER
Successes: hiPSC
Teruo Okano: ISSCR Yokohama 2012
…RPE regeneration…
Succsesses: hESC
Schwartz a kol. Lancet 2012; 379:713-20
Human embryonic stem cells
Pigment epithelium differenciation
Trounson, 2016
Shortlist of recent applications of PSC in clinical trials
(more info in part II – with Dr. Martin Pesl)
What is this for?
Regenerative and
reconstructive medicine
Disease modelling
Understanding
pathological processes
Drug development
Duchen Muscular
Dystrophy
-Affected sceletal muscles
-Heart failure
Hypothesis:
-Missing dystropin
-Sarcoplasmic reticulum
calcium leakage
-Would Ca2+ channel
inhibitors help?
We want to test it on
affected cells!!!
Found physiological differences but not severly
preventing natural heart muscle regeneration
and what would explain heart failure…
Duchen Muscular
Dystrophy
-Affected sceletal muscles
-Heart failure
Hypothesis:
-Missing dystropin
-Sarcoplasmic reticulum
calcium leakage
-Would Ca2+ channel
inhibitors help?
We want to test it on
affected cells!!!
0,00
0,20
0,40
0,60
0,80
1,00
1,20
Ratiofoci/nuclei
*WTDMD
spontaneo
us
MOX
WT DMD
**
***
*
D
CTRL MOX CTRL MOX0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
DMD WT
ROS
Relativefluorescence
DMDWT
*B
Duchen Muscular
Dystrophy
-Affected sceletal muscles
-Heart failure
Conclusion: defective dystrophin causes elevated ROS production
via NO synthase, causing SC mutagenesis and progenitor
depletion
Duchen Muscular
Dystrophy
-Affected sceletal muscles
-Heart failure
What is this for?
Regenerative and
reconstructive medicine
Disease modelling
Understanding
pathological processes
Drug development
Catecholinergic Polymorphic Ventricular Tachycardia (CPVT)
Sportment‘s Sudden Cardiac Death Syndrome
Marc Vivian Foe
Mutace RyR:
Pomalý únik Ca2+
Ca2+ channel inhibitor screen needed – will hiPSC help?
Classical proces impossible
Target organ biopsy
„ex vivo“/“in vitro“ cultivation
Putative drugs
We do not have access to
the sufficiently sized heart
biopsies
Skin biopsy
hiPSC
Embryonic bodies
… how to get heart cells of the patient who cannot
provide them..
α-actininp-cTnI
Patient specific hiPSC derived cardiomyocytes
Analysis using atomic force microscope
Biomechanical properties:
Skin biopsy
hiPSC
Embryonic bodies
… how to get heart cells of the patient who cannot
provide them..
Analysis using atomic force microscope
Biomechanical properties:
Skin biopsy
hiPSC
Embryonic bodies
… how to get heart cells of the patient who cannot
provide them..
Acimovic, Rotrekl a kol.
Conclusion: Succsessful stabilization of RyR2
and calstabin 2 binding by S107 compensated
CPVT fenotype.
Fluorescent microscopy
Skin biopsy
hiPSC
Embryonic bodies
… how to get heart cells of the patient who cannot
provide them..
Final discussion – PART I (pluripotent stem
cells)
•Ageing of the PSC in culture – dangerous genetic changes?
•Genetic reprogramming – is that a danger in iPS based therapy?
•Limitted diversity of PSC lines and associated GVH disease risk?
•Legislature sufficient/overcautios?