Induced pluripotent stem cells (IPs) 6 years to win the Nobel prize...

Transcription

1 Induced pluripotent stem cells (IPs) 6 years to win the Nobel prize...

2 induced Pluripotens Stem sejt (ipss) first publication in 2006 mouse / human, reprogramming embryonal / adult fibroblasts 2012: medical Nobel prize; Gurdon és Yamanaka

3 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)

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5 similar gene expression, methylation profile and telomerase activity in ES and ips cells chromatin immunprecipitation telomerase activity CpG methylation

6 global gene expression profile: ipscs are similar but not identical to ES cells global gene expression: DNA microarray heat map

7 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...)

8 retroviral transduction of Fbx15 bgeo/bgeo tail-tip adult fibroblasts (TTFs) by OSKM [+CAG-GFP expression] - gene expression is similar to embryonic clones pluripotency tests: - teratoma formation - tissue-specific differentiation of all 3 germ lines - blastocyst injection > chimera (GFP) but no tg offsprings

9 adult ipscs: - similar OSKM protein levels to ES, but Nanog level is lower RNA levels are increased - Oct4 and Sox2 levels decrease during in vtro differentiation - random transgene integration pattern - normal karyotype - spontaneous differentiation in the absence of feeder cells simple sequence length polymorphisms (SSLPs)

10 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; 5x10 5 fibroblasts -> 300 colonies - ES-like features: morphology, feeder dependence, gene expression and markers, CpG methylation, high telomerase activity, fast doubling time (~45h), EB formation

11 teratoma formation tissue-type differentiation: neuron, cardiac muscle, epithelium... germ cell production potential and tetraploid complementation was not tested 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 fibroblasts; 0,002%)

12 The new hype: ipscs! reprogramming! + Nanog and GFP expression (not necessary) Oct4, Sox2, Nanog, Lin28; pluripoteny tested by teratome formation

13 which factor(s) are needed really??

14 which factor(s) are needed really?? Myc - tumor-related factor - regulates HAT complexes: global histon acetylation -> decompacting chromatin -> allowing the action of Oct4 and Sox2 - > 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

15 Modifications necessary for human therapeutical useage of ipscs

16 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%)

17 piggybac transposon: seamless excision at the repeated ends (mutations???); transposon expression is only transiently needed teto promoter: inducible expression by doxycycline 2A virus genes: polycistronic; MKOS genes in a row excision happens at once problems: transposase, transient expression...

18 modified Epstein-Bar virus: orip/ebna1 vector - stable episomal (extrachromosomal) presence after selection <1% efficiency; 1 division/cell cycle - OCT4, SOX2, NANOG, LIN28, c-myc, KLF4 + SV40LT production in different ratios - in the lack of a proper selection, replication is hindered by mutations; early segregation (5% per cell cycles) - hipomethylated Oct4 and Nanog promoters; pluripotency, teratoma formation - final efficiency is very low: 3-6 colonies out of 10 6 cells

19 HIV-TAT 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..) very low efficiency (<0,001%) difficult task to produce sufficient amount and ratio of the inducing factors

20 Sendai virus: RNA based replication, so it can not integrate into the host genome - 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 (> cell cycles) surface HN antigen can be used to selectively remove virus-expressing cells optimalized protocol, but still very low efficiency

21 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 O 2 level, 1:1:3:1 K:M:O:S ratio <3 weeks; >2% efficiency...

22 pla-ipscs: episomal plasmid vectors, suppression of p53; expression of L- Myc (without transforming activity) can be differentiated towards dopaminergic neurons

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24 CiPSCs: chemically induced ipscs reprogramming by 7 small molecules Oct4-GFP mouse embryonic fibroblast (OG-MEF), viral expression of Sox2, Klf4, c-myc F: Forskolin (FSK), 2-methyl-5- hydroxytryptamine (2-Me-5HT), D4476: to evoke Oct4-mediated effects VC6T: VPA, CHIR99021 (CHIR), , Tranylcypromine besides Oct4 expression, it induces ips reprogramming VC6TF: increased GFP and E-cadherin expression, but Oct4 and Nanog promoters still hypermethylated DZNep [Z]: 3-deazaneplanocin A; screen in a DOX- Oct4 inducible expression system; increases Oct4 expression VC6TFZ: GFP expression is increased by 65x in OG-MEFs, reprogramming is not yet complete

25 2i: inhibiting glycogen synthase kinase 3 and MAPK signalling 1 month later ESC-like morphology, ips features 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

26 direct reprogramming to differentiated cells?

27 SunTag system: CRISPR activation (deactivated form of Cas9 fused with transactivation domains - promoting downstream gene transcription) single guide RNAs (sgrnas): target and activate Oct4 or Sox2 promoters / enhancers derepressing endogenous Oct4 or Sox2 expression is sufficient for ips formation and reprogramming via selected histone acetylation specific changes in the chromatin structure is sufficient to induce reprogramming

28 there are (still) many problems... Nature 471, (03 March 2011) doi: /nature09798 reprogramming: demethylation of CpG islands -> transcriptional activity methylation pattern is similar, but not identical between ips and ES cells: somatic memory? stochastic processes lead to interclonal differences large regions are resistant to demethylation, especially around the centrosome and the telomeres ES and ips cells are clearly NOT identical, raising the possibility of different developmental pathways

29 there are (still) many problems... Nature 474, (09 June 2011) doi: /nature10135 autolog ipss: in principle (?) these can not evoke immune response in the original source animal ViPSC: retrovirus-induced ips; EiPSC: episomally induced ips cells; transplantation into the original animals -> aberrant expression of tumor antigens (Hormad1, ZG16) T-cell dependent immune response: elimination of teratomes formed in syngenic animals 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

30 there are (still) many problems... epigenetic regulation of ips reprogramming : many similarities to early age carcinogenesis - unlimited self renewal - changes in the metabolism: increased importance of glicolysis - changes in the transcriptome - Myc, Klf4: oncogenes in certain somatic cells; Oct 3/4: increased expression in germline tumors enhanced selfrenewal? - preliminary termination of reprogramming often leads to tumor formation - reprogramming/cancer development is primarily directed by epigenetic factors and less by genetic mutations

31 Comparing reprogramming and differentiation similar processes happening in a converse order

32 Induced pluripotent stem cells (IPs) 6 years to win the Nobel prize and 8 years to commit a suicide

33 STAP (stimulus-triggered acquisition of pluripotency) cells

34 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

35 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

36 Therapeutic useage of ips technology important considerations

37 IPs in therapy important aspects main attempts: 1. cell/organ transplantation, tissue replacement 2. generation of disease models 3. patient-specific therapy, clinical trials

38 IPs in therapy important aspects main attempts: 1. cell/organ transplantation, tissue replacement 2. generation of disease models 3. patient-specific therapy, clinical trials

39 IPs in therapy important aspects main attempts: 1. cell/organ transplantation, tissue replacement 2. generation of disease models 3. patient-specific therapy, clinical trials 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) source of cells instead of donor fibroblasts: CD34+ umbilicar stem cells, T limphocytes? 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

40 Therapeutic useage of IPs generation of in vitro disease models - human-specific vs animal models - personalized medicine and screening source of pluripotent, diseasespecific cells - preimplantation genetical screening, affected embryos - in vitro mutagenesis of hes cell lines -ips generated from the somatic cells of the patients

41 Therapeutic useage of IPs generation of in vitro disease models: problems to solve - incomplete reprogramming: heterogenous cell populations - lack of standardized protocols - variability in genetic background - differences in epigenetic memory; X chromosome inactivation

42 Therapeutic useage of IPs

43 Therapeutic useage of IPs

44 IPs in therapy clinical trials and plans

45 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