Transcript trans-differentiation

“두려움 그것이 사람을 바보로 만든다”
Strategies of Regenerative
Medicine
실험
• 면역결핍생쥐의 등에 암세포를 이식하여
teratoma 형성 관찰
• 각 조별로 실험계획 작성
• - Title
• - Hypothesis
• - Objective
• - Materials and Methods(구체적으로)
• - Expected Results
• - Discussion
• - Reference
I. Introduction
• Four strategies for restoring tissue, organ and appendage
structure
1) The use of biomimetic (bionic) devices and organ
transplant: replacement
2) Cell transplantation: replacement
3) implantation of bioartificial tissues or organs: replacement
4) The chemical and physical inhibition of scarring and
simultaneous induction of new tissue formation directly a
the site of injury: true regeneration
• All 4 restores original function (paracrine, modulation injury,
etc), so they are all regeneration
II. Biomimetic devices and organ transplantation
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Biomimetic devices: hearing aids, joint replacements, cardiac valves, apendage prostheses
Limitation of total artificial heart
size (by age), mobility, duration of power source
Most difficult organ to mimic: liver and pancreas, because these organs perform complex
biochemical functions
Biomimetic devices have been developed, and will be developed.
Organ transplantation: developed from mid 20C
History
1901, Karl landsteiner, discover ABO blood antigen  blood transfusion
1954, Joseph Murray, first isogenic kidney transplant between identical twin brother
Allogenic organ transplantation was not success until 1980s by immune rejection 
development of anti-rejection drug “cyclosporin A”: immunosupressor
Today, most organs can be transplanted with high survival rate for all graft.
5 yr: 50%, 10 yr: 40%
the most spectacular event: face transplant
Shortage of organ donor is primary obstacles
Overcome of Immune rejection is hot issue without immunosupressor in allograft
Xenotransplantation using TG pig
problem of hyper-rejection by preformed antibodies to a sugar, α-1,3-galactose.
α-1,3-galactose residing on the surface of pig blood vessel endothelial cells
믿음이 (KO of α-1,3-galactose): still have rejection
III. Cell transplant
• Donor shortage can overcome through cell transplantation
• Preparing suspension cells  graft  aggregation to replace damaged
tissue
• Depends on the ability to harvest and expand cells to sufficient
numbers to replace damaged tissue
- for the cells to proliferate to the required number after
transplantation
- for the transplanted cells to develop into new tissue that becomes
integrated with the surrounding tissue
• Maintenance of normal tissue and regeneration of injured tissue
required biochemical signals from the initial stages of regeneration
• Transplanted cells must have the response ability to regeneration
• Often fibrosis suppress the regeneration signal of transplanted cells
• So, transplanted cells must have neutralization capacity of fibrosisinducing signals to the injury site, and regeneration-permissive
signaling molecules
A. Characteristics of the ideal cell for transplantation
• Cell source: central issue for cell transplantation
• Bone marrow stem cells: cure hematopoietic disease, but using
other cells (fetal midbrain cells for parkinson’s disease,
differentiated chondrocytes for defects in cartilage) have been
failed.
• The criteria of the ideal cells for transplant
1) be easy to access and harvest
2) be easy to expand to large numbers
3) have not senesced (노화)
4) be pluripotent: possess a developmental plasticity allowing it
to differentiate into any of the more than 200 cell types of
the human body
5) be rejection-proof
• To avoid immuno-rejection
- cells must be engineered to be immunologically inert
- so, the cells could be universal donor cells, or autogenic cells
B. Are adult stem cells pluripotent?
• Own Adult stem cells:
- ideal to regenerate tissues without rejection
- have a developmental potential much greater than their
prospective fate: trans-differentiation to beyond their
own lineage if transplanted into foreign niche
1.
Trans-differentiation of Bone Marrow stem cells (BMSC)
The primary clinical use in 1968 to regenerate hematopoietic system in
HLA(human leukocyte antigen)-identical siblings to treat immunedificiency and
leukemia
- expanded through tissue matching to unrelated donors and recipients
• BMSC is multipotential, having sufficient developmental plasticity to transdifferentiate into cell types in injured cells
• Undifferentiated BMC, purified population of HSC and MSCs
- reconstitute the hematopoietic system (the normal potential of HSC) in vivo
after injection into x-irradiated, scid, or PU.1-null mice
- trans-differentiated into cardiomyocytes, skeletal muscle, neurons, astrocytes,
hepatocytes, β-cells, kidney tubule epithelium, skin epithelia, lung, thymus,
intestine.
- most trans-differentiation rate is less than 1 ~ 4%, but 20% for lung
• Injection of Alpha-MHC promoter + Akt gene (GFP or c-myc tagged)
transgenic male BMC successfully formed cardiomyocytes, but did not mature
in female mice: research on precise differentiation factor is needed
- isolated cells in vitro after 15 – 30 days post-injection, labelled cells contracted
and exhibited electrical properties similar to the spared cardiomyocytes of the
infarct region
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Adult rat and human MSC trans-differentiated in vitro into neural-like cells induced by βmercaptoethanol, DMSO/butylated hydroxyanisole, RA, co-cultured with fetal midbrain cells
or postnatal hippocampal (해마회) astrocytes
- RA and Shh are important.
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Still in vitro results are not sufficient to apply to clinic
- mMSC transgenic for Lac-Z driven by the cardiac specific promoter (α-MHC) and ESCs to
form chimeric embryo bodies (EB) failed to differentiate into cardiomyocytes without EB
2. Transdifferentiation of non-marrow MSCs
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Satellite cells: small multipotent cells with no cytoplasm found in mature muscle
- precursors to skeletal muscle, give rise to satellite cells and differentiate to skeletal muscle
- Rat saterllite cells differentiated into NSCs cultured with neural differentiation medium, but
did not differentiation further
- satellite cells transplanted into normal heart transdifferentiated into cardiomyocytes
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Adipose derived stem cells (ADSCs: normally differentiated into adipocytes, osteoblasts,
chondrocytes, skeletal muscle) and subpopulation of dermal fibroblast
- transdifferentiated into cells expressing neuronal marker
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Heart, kidney, brain, skin tissue
- trans-differentiated into hepatocytes when transplanted into host liver
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Pericytes associated with capillary that respond to injury by becoming MSCs, pericytes
identified in multiple organ of the body
3. Trans-differentiation of epithelial stem cells
• Bone marrow stem cells
- transdifferentiated into myeloid and lymphoid blood cells in irradiated mice
• Neural stem cells, NSC
-differentiated into muscle when co-cultured with myoblasts, cardiomyocytes or ESC
• Rat liver stem cells
- transdifferentiated into cardiomyocytes when grafted into the myocardium, co-cultured
with neonatal rat or mice cardiomyocytes
- transdifferentiated into pancreatic islet
• Thymic epithelial stem cells (TECs)
- transdifferentiated into epidermal structure
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4. Transdifferentiation artifacts
Transdifferentiation often due to artifacts that give the illusion of transdifferentiation
induced by foreign niche signals
- contamination of injected test cells with other cells
- entry of host immune cells into an organ transplant where they are mistaken for
transdifferentiation
- fusion of donor cells with host cells to create a heterokaryon: express donor marker and
having 2 nuclei
- Incomplete transdifferentiation: donor cells may initially incorporated into foreign tissue
and express host cells molecular marker, but failed to complete reprogramming
C. Effects of Age on Adult regenerationcompetent cells
• Fibrosis and regeneration ability: declined by age
- aging, oxidative stress
- imbalance in intercellular level of free radical and cellular antioxidant
system
- imbalance reflects decreases in hair regeneration and turnover rate of
epidermis, etc.
• The questions for decline of regeneration capacity
- decline in the number of regeneration-competent cells
- decline in the intrinsic ability of cells to respond to regenerative needs
- a deteriorating niche environment
- some combination of above
• The answer for these question is crucial for all regenerative
medicine strategy based on the use of adult regenerationcompetent cells
• Parabiosis experiment in mice suggests that niche
deterioration is the primary culprit (범인) in loss of
regenerative capacity
1. liver hepatocytes
• partial hepatectomy
- young: well recover - C/EBPα level is reduced
- old: reduced proliferative response - C/EBPα level is not reduced
- mechanism:
liver transcription factor C/EBPα inhibits cdk (cyclin-dependent kinase)  form a
complex with TF (pRb, E2F) and Brm (chromatin remodeling protein)  growth
arrest of hepatocytes
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parabiosis experiment reversed the decline of liver regeneration in aged mice
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what is the major factor of this reverse ?
- transgenic animal experiment: not circulating cells.
2. Satellite cells of skeletal muscle
• Muscle regeneration is diminished by age
- the number of SCs is not diminished, but ability to proliferate declines
significantly by 25% compared to young
• Insufficient upregulation of the Notch ligand Delta and thus diminished
activation of Notch
- blocking Notch in young mice: marked inhibition of regeneration
- transplantation of aged muscle into young mice: regeneration capacity is
increased
- activation of Notch by antibody in aged mice: restores the regeneration
- parabiont with young-old mice: muscle regeneration of old mice restored
nearly to young mice
• The important factor is certain systemic niche factor
- in vitro experiment: treatment of young mice serum into cultured muscle cells
from old mice  upregulation of Delta expression  activation of Notch
signaling
• Muscles from old rat and human have deficiency of mechano growth factor
(MGH), a splice variant of IGF-I.
• Strength training restores the MGH production, particularly in combination with
GH administration
• Overall suggests that niche factor has a significant role on the ability of SCs in
aging muscle to express an undiminished regenerative potential
• 3. Hematopoietic Stem Cells
3. Hematopoietic Stem Cells
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Reduced capacity for expansion into myeloid and lymphoid progenitor cells in aged HSC
- self-renewal of LT (long term)-HSCs is not declined over the life of an animal
- ability of the transit amplifying populations to proliferate in response to stress is
diminished by accumulating deficiency in DNA repair mechanism
- heterchronic parabiosis experiment: niche is important to change in hematopoietic
decline as well.
- old serum significantly diminshed young HSCs expansion ability
- the factors in serum that change with age is not known, but mustly involve signaling.
4. Cardiovascular Stem and Progenitor Cells
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Circulating endothelial cell progenitor cells: continually repair the lining of blood vessel,
preventing plaque formation and atherosclerosis (동맥경화)
- the number of circulating endothelial stem cells (EnSCs) decreases with age in human
- relationship between the number of circulating EnSCs and risk for cardiovascular disease
- this relationship is a better predictor for cardiovascular disease than the standard set of
risk factor
- Bypass surgery patients showed the number of EsSCs and levels of VEGF, IL-6, IL-8, IL-10
reached significantly higher levels 6 hr after operation, but age over 69 did not.
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Cardiomyocytes were to die and be replaced by CSCs
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Cardiac stem cells (CSCs): decreased number and function by age: attenuated by apoptosis
until the loss of cardiomyocytes exceeded the number of new cardiomyocytes
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Differentiation and proliferation of CSCs regulated by IGF-I – PI3K – Akt pathway
5. Central nervous system stem cells
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Microarray data for functional cells in the aging human frontal cortex after age 40,
revealed that the downregulation of
- synaptic plasticity
- vesicular transport
- mitochondrial function
- stress response
- anti-oxidant related gene
- DNA repair gene
- overall related to DNA damage in the promoters of these genes
6. cyclin-dependent kinase inhibitor p16INK4a in stem cell aging
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During aging process: p16INK4a level is increased
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HSCs from TG mice deficient in p16INK4a maintained their repopulating capacity and less
cell death than aged counterpart.
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Overexpression of this gene in young mice: ???????
D. Embryonic Stem Cells (ESCs)
1. Derivation of ESCs
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Skip: everybody knows.
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Confirmation of pluripotency
- grafting them into blastocyst (except human
ESCs)
- grafting them into immunodeficiency mice
to form teratoma
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mESCs form domed colony with LIF acting
through STAT3 pathway
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BMP4 work concert with LIF, acting as an
inhibitors of differentiation
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mESCs maintained with LIF, wo serum and
feeder cells by stimulating Wnt pathway +
ERK1/2 and GSK3 inhibitors
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hESCs form flat colonies need FGF2, activin,
Lefty2 and IGF signaling for maintenance
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hESCs are not naïve (undifferentiated) as
mESCs: currently not available to culture naïve
condition
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Epigenetic basis of pluripotency
ESCs can proliferate ad infinitum while remaining pluripotency
Set of TF
- human and mice: Oct4, Sox2, Nanog, Tcf3, Klf4, c-Myc, ESRRB, Sall4, Tbx3,
STAT3
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In the developing mice embryo, pluripotent cells become restricted to epiblast:
self-renewing and pluripotent until gastrulation
• Cells from epiblast are more primed to differentiation (less naïve) than ESCs:
different growth property and TF signals
The epigenetic regulatory circuitry
- derivation: ICM from zygote
- morula to blastocyst: chromatin undergoes epigenetic modification
 activating the genes for pluripotent of ICM
 repressing the genes for differentiation
• The modification
- addition or subtraction of acety or methyl groups to specific AA of histones
- methylation or demethylation of the DNA itself at CpG (cytidine-phosphateguanine) islands
- by histone and DNA methyltransferase, demetylases, histone acetyltransferase,
deacetylase
- combination of these: determine the degree of compaction of the chromatin
and transcriptional activity
- Pluripotency gene: hypomethylated; differentiation: hypermethylated
- hESCs vs. fetal lung fibroblast: histone acetylation and methylation, DNA
methylation are dramatically changed
• Micro RNA (miRNA)
- associated with repression of differentiation gene
- ESc deficient in miRNA processing enzyme: abnormal proliferation and defects
in differentiation
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The mechanism for silencing of pluripotent genes and activation of differentiation genes
during the transition from pluripotent to differentiation: do not KNOW
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3. Directed Differentiation of ESCs
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Withdrawal of GF  EB (embrioid body) formation
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EB contains precursor cells for 3 germ layers
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Transferring EB to differentiation culture medium: differentiated into specific cell type
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Direct differentiation: neurons, glia or their precursors, neural crest stem cells, retinal
pigment epithelium, rod and cone photoreceptors, lens and neural retina cells,
keratinocytes, cardiomyocytes, osteoblasts and lung alveolar epithlium, male gametes,
endothelial cells, hematopoietic precursors including hemangioblast, mature T-cells
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Differentiation factors: some combination of small molecules – RA
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endoderm and endocrine cells: IDE1 and 2
4. Risks Associated with transplantation of ESC derivatives
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ESCs as a prime source for transplantation of cell therapy
- expanded indefinitely in culture
- differentiatable to every cell type
- having zero senescence status
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Potential risks
- chromosome abnormalities induced by culture: deletion, duplication, amplification
- transplanted ESCs can form tumor
- incorrect differentiation, inappropriate localization, produce excessive number of
progeny cells
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each lineage has different differentiation ablility
5. “Designer” ESCs from SCNT Blastocyst
• To overcome immunoreject of transplanted ESCs: patient-specific (autogenic) ESCs from
somatic cells (SCNT technology) are solution
- a technique pioneered by Gourdon in amphibian (1975) and Wilmut and Campbell in
mammals (2002)
- No need to explain because everybody knows the mechanism
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dESCs (designer ESCs)
- first created for mice and be capable to differentiating into
neurons and muscle in vitro
- dESCs successful to fertile adult mice: having pluripotency
- differentiate into three germ layers
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Three drawbacks to the creation of human dESCs by SCNT
1) shortage of human egg
2) low efficiency of reprogramming
3) ethics and morality
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Using animal eggs as the recipient for human nuclei
Chen et al (2003)
- fusing enucleated rabbit egg with human somatic nuclei
- successful derivation of hESCs
- growth potential is not clear
- become unstable due to impaction between human nuclear gene and rabbit
mitochondrial gene
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Possible solution: human induced pluripotent stem cells
- however equivalent ability is still questions.
E. Induced Pluripotent Stem Cells (iPSCs)
1. Reprogramming of Somatic cells to iPSCs
• 2006, Takahashi Yamanaka used a combination of four ESC pluripotent TF: OCT4, SOX2,
KLF4, c-MYC (OSKM)
• Reprogrammed mouse dermal fibroblast to pluripotent stem cells with characteristics of
ESCs
• 2007, Takahashi and Yu develop human iPSCs.
- Takahashi used OSKM
- Yu used OSLN (Oct4, Sox2, Lin28, Nanog)
• Comparative analysis between ESCs and iPSCs
- similar morphology and growth characteristics
- global patterns of gene activity
- DNA methylation status of
pluripotency gene promoter
Successfully reprogrammed mice iPSCs
- reactivation of pluripotency gene
- inactivation of X chromosome genes in female cells
- upregulated telomerase
- differentiated into all three germ layers in the chimeric embryo assay and after
intramuscular injection into SCID mice
- generate a complete mice when injected into blastocyst of tetraploid embryo
• * tetraploid technique
- fusion of two blastomere  produce tetraploid blastocyst  develop a trophoblast, not
a ICM.
- injected diploid iPSCs develop to epiblast
• Take home task to overcome
1) low efficiency
- partial reprogramming by incomplete chromatin modification
- repression of differentiation-specific transcription factor
2) vector integration of genome
- virus and delivery vector is integrated into host genome
- influence differential potential or even cause malignancy, c-Myc (protooncogene)
3) Techniques must developed
- more easier and efficient methods
- minimize the number of TF for reprogramming
- decrease reliance on retroviral vectors, increase efficiency by simultaneous treatment with
a number of small molecules
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2. Directed Differentiation of iPSCs
• Use sets of growth factors, substrates and other factors known to drive specific cell
differentiation from stem cells and progenitors during development
3. Advances in Reprogramming methods
• To improve the speed and efficiency of reprogramming while simultaneously eliminating
exogenous DNA from the induction process
1) First step: Selection of cell types other than fibroblast
- mHepatocytes and Gastric epithelial cells, m and hB-lymphocytes, hT-cells, hTestis cells,
hCord blood-derived endothelial cells have been reprogrammed to iPS
- human keratinocyte: much easier to reprogram with OSKM: takes half as long as
fibroblast, 100 fold more efficient
- hepatocytes and gastric epithelium: need only a few number of retrovirus integration
sites, thus do not cause tumor formation in chimeric mice
2) Next step: simplify the induction mix
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2) Next step: simplify the induction mix
- c-myc is not necessary for low-efficiency reprogramming
- Oct4 and Sox2 are core pluripotency gene
- Without KMN or L, cells are capable to reprograme, but reprogramming is delayed
and efficiency is greatly reduced, suggest that these factors are supporting genes
Some of the small molecules
: enhance reprogramming by inhibitory effects on methylation or deacetylation
- histone deacetylase 2 inhibitor, valproic acid (VPA)
Transfection with OSK, 100-fold increase reprogramming efficiency in MEF (mouse
embryonic fibroblast)
Promoting reprogramming efficiency transfected with OS in human fibroblast
Oct4 can be replaced to Nr5a2, which shares many common target genes: Sox2, Klf4,
Nanog
VitC: enhances reprogramming efficiency in mouse and human fibroblast transfected with
OSK or OSKM
Two TGF-β receptor 1 kinase inhibitors (E-616452, E-616451) can substitute Sox2 in OSK
triad, indicating that TGF-β signaling is inimical to differentiation
Oct4 alone can reprogrme in human and mouse neural stem cells, intrinsically activate
Sox2 and c-Myc with 0.004-0.006% of low efficiency
- Oct 4 transfection with small molecule PS48 (activator of PDK1), BayB, A-83-01, parnate,
and CHIR99021 able to reprograme keratinocytes, human umbilical vein endothelial
cells, adipose derived stem cells to iPSCs
Retroviral transfection of the TF (Tbx3) with OSK into MEF produced iPSCs with improved
germ-line competency
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Elimination the problem of viral integration
non-integrating adenovirus, lentivirus and plasmids used as vector for OSKM
OSKM proteins themselves fused to a cell membrane penetrating peptide, since the
proteins degraded over 48 hours
proteins introduced with VPA, efficiency was elevated to 0.006% for OSKM and 0.002% for
OSK
microRNA can be used as a reprogramming agent
Transfection of the miRNA cluster miR302/367 into mouse and human fibroblast in the
presence of VPA: 2 times greater than OSKM transfection
Introduction of synthetic mRNA for OSKM
Above all methods are tremendous advance for generating iPSCs in time and cost effective
5. Clinical issues with iPSCs
Hampers to clinical application
- these methods can achieve 100 % differentiation without teratocarcinoma formation
- whether iPSCs will differetiate as stably and diversely as ESCs
- long treatment time
For the clinical use of iPSCs: establishment  expanding  differentiating and expanding
precursor cells  testing teratoma: over two years, to slow to treat case like acute spinal
cord injuries
- Cost of production is high
Several times more expensive than patient-specific skin grafts ($100,000)
Solution: allogenic iPSCs bank with database of large population of immunological match
IV. Bioartificial tissue construction
A. Types of Bioartificial Tissues