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induced Pluripotens Stem sejt (iPSs)
• first publication in 2006
• mouse / human, reprogramming embryonal / adult fibroblasts
• 2012: medical Nobel prize; Gurdon és Yamanaka
• FBx15: ES specific expression, but not essential for pluripotency
• reporter: b galactosidase + neomycin resistence driven by the FBx15 promoter; weaker resistance in somatic cells
• retroviral transduction of 24 factors: formation of ES-like cells (EBs, methylation profiles)
CpG demethylation
• selecting the critical 4 factors (Oct4, Sox2, c-Myc, Klf4 - Yamanaka faktors, OSKM)
• similar gene expression, methylation profile and telomerase activity in ES and iPS cells
chromatin immunprecipitation
CpG methylation telomerase activity
• global gene expression profile: iPSCs are similar but not identical to ES cells
global gene expression: DNA
microarray – heat map
• pluripotency tests:
- teratoma formation
- tissue-specific differentiation of all 3 germ lines in vivo and in vitro (majority of iPS clones)
- formation of embryoid bodies (EBs)
(chimera formation, germ cell production potential and tetraploid complementation was not tested...)
• retroviral transduction of Fbx15bgeo/bgeo tail-tip adult fibroblasts (TTFs) by OSKM [+CAG-GFP expression]
• pluripotency tests:
- teratoma formation
- tissue-specific differentiation of all 3 germ lines
- blastocyst injection –> chimera (GFP) but no tg offsprings
- gene expression is similar to embryonic clones
• adult iPSCs:
- random transgene integration pattern
- normal karyotype
- spontaneous differentiation in the absence of feeder cells
- similar OSKM protein levels to ES, but Nanog level is lower – RNA levels are increased
simple sequence
length polymorphisms
(SSLPs)
- Oct4 and Sox2 levels decrease during in vtro differentiation
• same 4 OSMK factors, but the protocol is optimized for human cells - viral transduction efficiency is increased by the mouse RV
receptor; bFGF dependence
- slower progress; 5x105 fibroblasts -> 300 colonies
- ES-like features: morphology, feeder dependence, gene expression and markers, CpG methylation, high telomerase activity, fast doubling time (~45h), EB formation
• teratoma formation
• tissue-type differentiation: neuron, cardiac muscle, epithelium...
• retrovirus (and normal) OSKM expression is strongly silenced during differentiation
• problems: 3-6 integration sites / retrovirus -> increased tumorigenesis? (mouse iPS: >20% of mice derived from iPS developed tumors)
• problems: despite retroviral transduction, low efficiency (10 iPS clones from 50.000 fibroblasts; 0,002%)
• germ cell production potential and tetraploid complementation was not tested
• + Nanog and GFP expression (not necessary)
• Oct4, Sox2, Nanog, Lin28; pluripoteny tested by teratome formation
The new hype: iPSCs! reprogramming!
which factor(s) are needed really?? Myc
- tumor-related factor
- >25.000 c-Myc binding sites
- c-Myc is tumorigenic, L-Myc is more potent
Oct3/4, Sox2 - highly expressed in ES cells/early embriogenesis: maintaining
pluripotency
Klf4 - maintenance of ES phenotype and proliferation
- repressing p53 functions, which supresses Nanog -> Nanog activation
+ Lin28 - promoting Oct3/4 production; increasing the efficiency of viral
transduction
+ SALL4, Esrrb - partly substituting Klf4- mediated effects; increasing the efficiency
of reprogramming
- regulates HAT complexes: global histon acetylation -> decompacting chromatin -> allowing the action of Oct4 and Sox2
• c-Myc: strong tumorigenic effect, but reprogramming efficiency is very low without it
• 2 vectors: 1) Oct3/4, Sox2 and Klf4 coded by a polycistronic plasmid; in a given order; 2) c-Myc is encoded on a separate plasmid
• multiple, repeated transfections in Nanog-GFP cells
• no (?) genomic integration, but very low efficiency ( <0,0002%)
• piggyBac transposon: seamless excision at the repeated ends (mutations???); transposon expression is only transiently needed
• 2A virus genes: polycistronic; MKOS genes in a row – excision happens at once
• tetO promoter: inducible expression by doxycycline
• problems: transposase, transient expression....
• modified Epstein-Bar virus: oriP/EBNA1 vector
- stable episomal (extrachromosomal) presence after selection – <1% efficiency; 1 division/cell cycle
- in the lack of a proper selection, replication is hindered by mutations; early segregation (5% per cell cycles)
- OCT4, SOX2, NANOG, LIN28, c-Myc, KLF4 + SV40LT production in different ratios
- final efficiency is very low: 3-6 colonies out of 106 cells
- hipomethylated Oct4 and Nanog promoters; pluripotency, teratoma formation
• HIV-TAT 48-60 as: many basic (Arg/Lys) aa [CPP], can penetrate the plasma membrane; within the nucleus, it regulates gene expression
• individual factors are produced by HEK293 cells; fibroblasts are „infected” by these lysates: p-hiPS (protein induced human iPSCs)
• very slow process (~80 days, multiple treatments are needed..)
• difficult task to produce sufficient amount and ratio of the inducing factors
• very low efficiency (<0,001%)
• Sendai virus: RNA based replication, so it can not integrate into the host genome
• optimalized protocol, but still very low efficiency
- DF: lack of protein F, so it is incapable of spontaneous infections/replications
• due to the differences in the replication speed between the virus and the host cell, the virus is gradually lost from the host cells (> 60-80 cell cycles)
• surface HN antigen can be used to selectively remove virus-expressing cells
• RiPSCs: RNA induced pluripotent stem cells
• repeated treatments with synthetic mRNAs + avoiding innate antiviral defence mechanisms
- 5-methylcytidine, pseudouridine: modified ribonucleoside bases
- attenuated interferon activation: reduced innate reactions
• nucleus-targeted, transient expression (12-18h) is satisfactory
• low O2 level, 1:1:3:1 K:M:O:S ratio
• <3 weeks; >2% efficiency.....
• pla-iPSCs: episomal plasmid vectors, suppression of p53; expression of L-Myc (without transforming activity)
• can be differentiated towards dopaminergic neurons
• Oct4-GFP mouse embryonic fibroblast (OG-MEF), viral expression of Sox2, Klf4, c-Myc
• CiPSCs: chemically induced iPSCs – reprogramming by 7 small molecules
• VC6T: VPA, CHIR99021 (CHIR), 616452, Tranylcypromine – besides Oct4 expression, it induces iPS reprogramming
• VC6TFZ: GFP expression is increased by 65x in OG-MEFs, reprogramming is not yet complete
• DZNep [Z]: 3-deazaneplanocin A; screen in a DOX-Oct4 inducible expression system; increases Oct4 expression
• VC6TF: increased GFP and E-cadherin expression, but Oct4 and Nanog promoters still hypermethylated
• F: Forskolin (FSK), 2-methyl-5-hydroxytryptamine (2-Me-5HT), D4476: to evoke Oct4-mediated effects
• TTNBP: synthetic retinoid acid analogue; 40x reprogramming efficiency
• reprogramming of neonatal and adult mouse fibroblasts and adipocytes, without the OG transgenic background
• pluripotency: mouse chimeras
• better survival in the lack of c-Myc expression, no tumor formation
• 2i: inhibiting glycogen synthase kinase 3 and MAPK signalling 1 month later – ESC-like morphology, iPS features
• SunTag system: CRISPR activation (deactivated form of Cas9 fused with transactivation domains - promoting downstream gene transcription)
• derepressing endogenous Oct4 or Sox2 expression is sufficient for iPS formation and reprogramming via selected histone acetylation
• single guide RNAs (sgRNAs): target and activate Oct4 or Sox2 promoters / enhancers
• specific changes in the chromatin structure is sufficient to induce reprogramming
there are (still) many problems.... Nature 471, 68–73 (03 March 2011)
doi:10.1038/nature09798
• reprogramming: demethylation of CpG islands -> transcriptional activity
• large regions are resistant to demethylation, especially around the centrosome and the telomeres
• methylation pattern is similar, but not identical between iPS and ES cells: somatic memory?
• stochastic processes lead to interclonal differences
• ES and iPS cells are clearly NOT identical, raising the possibility of different developmental pathways
Nature 474, 212–215 (09 June
2011) doi:10.1038/nature10135
• autolog iPSs: in principle (?) these can not evoke immune response in the original source animal
• T-cell dependent immune response: elimination of teratomes formed in syngenic animals
• ViPSC: retrovirus-induced iPS; EiPSC: episomally induced iPS cells; transplantation into the original animals -> aberrant expression of tumor antigens (Hormad1, ZG16)
• CD4-/- or CD8-/- mice do not show teratome elimination as both Tcell pools are needed
• conclusion: prior to clinical useage, the state (and useability) of the iPS cells must be checked
there are (still) many problems....
• epigenetic regulation of iPS „reprogramming” : many similarities to early age carcinogenesis
- unlimited self renewal
- changes in the transcriptome
- Myc, Klf4: oncogenes in certain somatic cells; Oct 3/4: increased expression in germline tumors – enhanced self-renewal?
- preliminary termination of reprogramming often leads to tumor formation
- reprogramming/cancer development is primarily directed by epigenetic factors and less by genetic mutations
there are (still) many problems....
- changes in the metabolism: increased importance of glicolysis
Induced pluripotent stem cells (IPs)
– 6 years to win the Nobel prize...
... and 8 years to commit a suicide
STAP cells
• simple protocol: generation of iPS cells from any source depending on mechanical dissociation and an acidic buffer ( pH=5,7, 30 min)
• even trophoblasts cells are formed?
• retracted in 5 months https://ipscell.com/2014/09/guestpostzubinmaster/
STAP cells
• simple protocol: generation of iPS cells from any source depending on mechanical dissociation and an acidic buffer ( pH=5,7, 30 min)
• even trophoblasts cells are formed?
• retracted in 5 months
IPs in therapy – important aspects
• main attempts: 1. cell/organ transplantation, tissue replacement 2. generation of disease models 3. patient-specific therapy, clinical trials
IPs in therapy – important aspects
• main attempts: 1. cell/organ transplantation, tissue replacement 2. generation of disease models 3. patient-specific therapy, clinical trials
IPs in therapy – important aspects
• in many cases, metabolic problems restrict iPS technology – useage of hES cells or need for allogenic iPSC cell banks (based on HLA types from healthy donors)
• is there a reliable and reproducible protocol for complete tissue-like differentiation? xeno-free culture protocols? foreign genomes?
• in vitro artefacts/mutations during the cultivation period (?)
• how to model late onset diseases – speeding up aging?
• business matters: copyright, royalty, pattern vs sharing the information
• main attempts: 1. cell/organ transplantation, tissue replacement 2. generation of disease models 3. patient-specific therapy, clinical trials
• source of cells instead of donor fibroblasts: CD34+ umbilicar stem cells, T limphocytes?
• generation of in vitro disease models
- human-specific vs animal models
• source of pluripotent, disease-specific cells
- preimplantation genetical screening, affected embryos
- in vitro mutagenesis of hES cell lines
-iPS generated from the somatic cells of the patients
Therapeutic useage of IPs
- personalized medicine and screening
• generation of in vitro disease models: problems to solve
- incomplete reprogramming: heterogenous cell populations
Therapeutic useage of IPs
- lack of standardized protocols
- variability in genetic background
- differences in epigenetic memory; X chromosome inactivation
IPs in therapy – clinical trials
• 2014, Japan: 77 years old woman, autologous iPS->RPE transplantation
• some improvements, no tumor – but point mutations discovered (due to aging?)
• 2017, Japan: transplantation of donor-derived RPEs; importance of cell banking
https://www.nature.com/news/japanese-man-is-first-to-receive-reprogrammed-stem-cells-from-another-person-1.21730
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