Induced pluripotent stem cells (IPs)

– 6 years to win the Nobel prize...

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??

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

Modifications necessary for human

therapeutical useage of iPSCs

• 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

• direct reprogramming to differentiated cells?

• 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

Comparing reprogramming and differentiation

• similar processes happening in a converse order

Induced pluripotent stem cells (IPs)

– 6 years to win the Nobel prize...

... and 8 years to commit a suicide

STAP (stimulus-triggered acquisition of pluripotency) cells

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

Therapeutic useage of iPS technology –

important considerations

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

Therapeutic useage of IPs

Therapeutic useage of IPs

IPs in therapy – clinical trials and plans

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