Reprogramming of Fibroblasts to Human iPSCs by CRISPR Activators

CRISPR-mediated gene activation (CRISPRa) can be used to target endogenous genes for activation. By targeting pluripotency-associated reprogramming factors, human fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSCs). Here, we describe a method for the derivation of iPSCs from human fibroblasts using episomal plasmids encoding CRISPRa components. This chapter also provides procedure to assemble guide RNA cassettes and generation of multiplexed guide plasmids for readers who want to design their own guide RNAs.Peer reviewe

Novel Approaches for Pluripotent Reprogramming

Somatic cells can be reprogrammed to pluripotent state by ectopic expression of a defined set of transcription factors. These induced pluripotent stem cells (iPSC) hold great potential for biomedical applications, such as disease modelling, drug discovery and cell therapies. The derivation of iPSCs is a complex multistep process that can commonly result in inefficient or incomplete conversion of the cells. The reprogramming efficiency and the quality of the reprogrammed cells can be affected by various components of the reprogramming method, including reprogramming vectors, starting cell populations and the choice of reprogramming factors. The aim of this thesis was to explore novel approaches for improving the pluripotent reprogramming outcome. The particular aims of this thesis were to investigate the use of recombinant Adeno-associated virus (rAAV) as a gene transfer vector for cellular reprogramming, characterization of the effects of old donor age and long term passaging on the reprogramming of fibroblasts, and development of reprogramming methods based on CRISPR/Cas9-mediated activation of endogenous reprogramming factors. In this study, rAAV-mediated transduction of mouse embryonic fibroblasts with OCT4, SOX2, KLF4 and C-MYC was found to successfully induce reprogramming to pluripotency. Unlike initially expected, the AAV vectors were integrated with high efficiency into the host genome during the reprogramming process, resulting in all analyzed iPSCs containing vector integrations. Both donor age and the culture time of the donor fibroblasts correlated with reduction in pluripotent reprogramming efficiency. This effect was found to be associated with upregulation of cellular P21 expression and reduction in cell proliferation. Downregulation of P21 expression by siRNA treatment was able to promote reprogramming of late passage senescent fibroblasts. By optimizing the reprogramming factor guide composition, CRISPR/Cas9-mediated gene activation (CRISPRa) could be used to derive iPSCs reprogrammed fully by targeted activation of endogenous genes. The efficient reprogramming of somatic cells by CRISPRa was found to be dependent on the inclusion of additional guides targeting an embryonic genome activation enriched Alu-motif. Due to the direct targeting of endogenous loci and the high multiplexing capacity of CRISPRa, the reprogramming approach has a high potential for mediating comprehensive and specific reprogramming. Overall, this thesis provides a number of novel tools and insights into the pluripotent reprogramming process. The results of this work can be used to develop more robust reprogramming methods and to improve the quality of reprogrammed cells.Erilaistuneita soluja voidaan uudelleenohjelmoida monikykyiseen (pluripotenttiin) tilaan ilmentämällä niissä määrättyjä geeninsäätelytekijöitä. Nämä indusoidut pluripotentit kantasolut (iPS-solut) ovat hyödyllisiä erilaisiin biolääketieteellisiin sovelluksiin, kuten tautimallinnukseen, lääkeainekehitykseen sekä mahdollisiin soluterapioihin. iPS-solujen ohjelmointi on monivaiheinen ja monimutkainen prosessi, joka saattaa johtaa solujen tehottomaan tai epätäydelliseen muuntautumiseen takaisin kantasolutilaan. Monet ohjelmointimenetelmien osat saattavat vaikuttaa ohjelmoinnin tehokkuuteen tai ohjelmoitujen solujen laatuun. Näihin kuuluvat muun muassa käytetyt geeninsiirtovektorit, ohjelmoitavat lähtösolupopulaatiot tai ohjelmointiin käytettävät geeninsäätelytekijät. Tämän tutkimuksen päämääränä on ollut etsiä uusia menetelmiä, joilla uudelleenohjelmoinnin lopputuloksia saadaan parannettua. Tarkemmat tavoitteet väitöskirjatyölle ovat olleet: tutkia rekombinantin Adeno assosioidun viruksen (rAAV) käyttöä geeninsiirtovektorina solun uudelleenohjelmointia varten, selvittää ikääntymisen ja pitkäaikaisen soluviljelyn vaikutukset fibroblastien ohjelmointiin sekä kehittää solun omien geenien aktivaatiota hyödyntäviä CRISPR/Cas9-pohjaisia menetelmiä solujen uudelleenohjelmointiin. Hiiren alkion fibroblastien transduktio rAAV pohjaisilla OCT4, SOX2, KLF4 ja MYC siirtogeeneillä johti onnistuneesti pluripotenttien solujen ohjelmointiin. Toisin kuin alun perin odotettiin, AAV vektorit integroituivat tehokkaasti ohjelmoitumisen aikana kohdesolujen genomiin ja kaikista tutkituissa iPS-soluklooneissa löytyi genomisia AAV vektorien sekvenssejä. Sekä luovuttajan korkea ikä, että pitkäkestoinen soluviljely laskivat uudelleenohjelmointitehokkuutta. Tämä ilmiö liittyi kohonneeseen P21 geenin ilmentymiseen sekä hidastuneeseen solunjakautumiseen. P21 ilmenemistason laskeminen siRNA välitteisesti edisti pitkään viljeltyjen hitaasti kasvavien solujen ohjelmointia. Optimoimalla uudelleenohjelmointigeenien aktivaatiota kohdentavien opas-RNA molekyylien koostumusta, saatiin CRISPR/Cas9 välitteisellä geeniaktivaatiolla (CRISPRa) aikaan iPS-soluja. Tehokas erilaistuneiden solujen uudelleenohjelmointi oli riippuvainen ylimääräisistä opas-RNA molekyyleista, jotka kohdensivat alkion genomin aktivaation alueille rikastunutta Alu-sekvenssi. Koska CRISPRa:lla saa kohdennettua suurella kapasiteetilla suoraan solun omia geenejä aktivoitavaksi, menetelmällä on varteenotettava mahdollisuus välittää tehokkaasti ja kokonaisvaltaisesti solutilan tarkkaa uudelleenohjelmointia. Kaikkiaan tämä väitöskirjatyö tarjoaa monia uusia työkaluja ja näkemyksiä solujen uudelleenohjelmointiin. Tämän työn tuloksia voidaan käyttää yhä luotettavampien ohjelmointimenetelmien kehittämiseen sekä ohjelmoitujen solujen laadun parantamiseen

Generation of iPSC line HEL47.2 from healthy human adult fibroblasts

Human iPSC line HEL47.2 was generated from healthy 83-year old male dermal fibroblasts using non-integrative reprogramming method. Reprogramming factors Oct3/4, Sox2, Klf4, and cMyc were delivered using Sendai viruses. (C) 2015 Elsevier B.V. All rights reserved.Peer reviewe

Generation of iPSC line HEL24.3 from human neonatal foreskin fibroblasts

Human iPSC line HEL24.3 was generated from healthy human foreskin fibroblasts using non-integrative reprogramming method. Reprogramming factors Oct3/4, Sox2, Klf4, and cMyc were delivered using Sendai viruses. (C) 2015 Elsevier B.V. All rights reserved.Peer reviewe

Combined negative effect of donor age and time in culture on the reprogramming efficiency into induced pluripotent stem cells

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSC) by the forced expression of the transcription factors OCT4, SOX2, KLF4 and c-MYC. Pluripotent reprogramming appears as a slow and inefficient process because of genetic and epigenetic barriers of somatic cells. In this report, we have extended previous observations concerning donor age and passage number of human fibroblasts as critical determinants of the efficiency of iPSC induction. Human fibroblasts from 11 different donors of variable age were reprogrammed by ectopic expression of reprogramming factors. Although all fibroblasts gave rise to iPSC colonies, the reprogramming efficiency correlated negatively and declined rapidly with increasing donor age. In addition, the late passage fibroblasts gave less reprogrammed colonies than the early passage cell counterparts, a finding associated with the cellular senescence-induced upregulation of p21. Knockdown of p21 restored iPSC generation even in long-term passaged fibroblasts of an old donor, highlighting the central role of the p53/p21 pathway in cellular senescence induced by both donor age and culture time. (C) 2015 The Authors. Published by Elsevier B.V.Peer reviewe

Generation of a SOX2 reporter human induced pluripotent stem cell line using CRISPR/SaCas9

SOX2 is an important transcription factor involved in pluripotency maintenance, pluripotent reprogramming and differentiation towards neural lineages. Here we engineered the previously described HEL24.3 hiPSC to generate a SOX2 reporter by knocking-in a T2A fused nuclear tdTomato reporter cassette before the STOP codon of the SOX2 gene coding sequence. CRISPR/SaCas9-mediated stimulation of homologous recombination was utilized to facilitate faithful targeted insertion. This line accurately reports the expression of endogenous SOX2 and therefore constitutes a useful tool to study the SOX2 expression dynamics upon hiPSC culture, differentiation and somatic cell reprogramming. (C) 2017 The Authors. Published by Elsevier B.V.Non peer reviewe

Conditionally Stabilized dCas9 Activator for Controlling Gene Expression in Human Cell Reprogramming and Differentiation

CRISPR/Cas9 protein fused to transactivation domains can be used to control gene expression in human cells. In this study, we demonstrate that a dCas9 fusion with repeats of VP16 activator domains can efficiently activate human genes involved in pluripotency in various cell types. This activator in combination with guide RNAs targeted to the OCT4 promoter can be used to completely replace transgenic OCT4 in human cell reprogramming. Furthermore, we generated a chemically controllable dCas9 activator version by fusion with the dihydrofolate reductase (DHFR) destabilization domain. Finally, we show that the destabilized dCas9 activator can be used to control human pluripotent stem cell differentiation into endodermal lineages.Peer reviewe

Kaposi’s Sarcoma-Associated Herpesvirus Reactivation by Targeting of a dCas9-Based Transcription Activator to the ORF50 Promoter

CRISPR activation (CRISPRa) has revealed great potential as a tool to modulate the expression of targeted cellular genes. Here, we successfully applied the CRISPRa system to trigger the Kaposi’s sarcoma-associated herpesvirus (KSHV) reactivation in latently infected cells by selectively activating ORF50 gene directly from the virus genome. We found that a nuclease-deficient Cas9 (dCas9) fused to a destabilization domain (DD) and 12 copies of the VP16 activation domain (VP192) triggered a more efficient KSHV lytic cycle and virus production when guided to two different sites on the ORF50 promoter, instead of only a single site. To our surprise, the virus reactivation induced by binding of the stable DD-dCas9-VP192 on the ORF50 promoter was even more efficient than reactivation induced by ectopic expression of ORF50. This suggests that recruitment of additional transcriptional activators to the ORF50 promoter, in addition to ORF50 itself, are needed for the efficient virus production. Further, we show that CRISPRa can be applied to selectively express the early lytic gene, ORF57, without disturbing the viral latency. Therefore, CRISPRa-based systems can be utilized to facilitate virus–host interaction studies by controlling the expression of not only cellular but also of specific KSHV genes

Kaposi’s Sarcoma-Associated Herpesvirus Reactivation by Targeting of a dCas9-Based Transcription Activator to the ORF50 Promoter

CRISPR activation (CRISPRa) has revealed great potential as a tool to modulate the expression of targeted cellular genes. Here, we successfully applied the CRISPRa system to trigger the Kaposi’s sarcoma-associated herpesvirus (KSHV) reactivation in latently infected cells by selectively activating ORF50 gene directly from the virus genome. We found that a nuclease-deficient Cas9 (dCas9) fused to a destabilization domain (DD) and 12 copies of the VP16 activation domain (VP192) triggered a more efficient KSHV lytic cycle and virus production when guided to two different sites on the ORF50 promoter, instead of only a single site. To our surprise, the virus reactivation induced by binding of the stable DD-dCas9-VP192 on the ORF50 promoter was even more efficient than reactivation induced by ectopic expression of ORF50. This suggests that recruitment of additional transcriptional activators to the ORF50 promoter, in addition to ORF50 itself, are needed for the efficient virus production. Further, we show that CRISPRa can be applied to selectively express the early lytic gene, ORF57, without disturbing the viral latency. Therefore, CRISPRa-based systems can be utilized to facilitate virus–host interaction studies by controlling the expression of not only cellular but also of specific KSHV genes

MASTL is enriched in cancerous and pluripotent stem cells and influences OCT1/OCT4 levels

Publisher Copyright: © 2022 The Author(s)MASTL is a mitotic accelerator with an emerging role in breast cancer progression. However, the mechanisms behind its oncogenicity remain largely unknown. Here, we identify a previously unknown role and eminent expression of MASTL in stem cells. MASTL staining from a large breast cancer patient cohort indicated a significant association with β3 integrin, an established mediator of breast cancer stemness. MASTL silencing reduced OCT4 levels in human pluripotent stem cells and OCT1 in breast cancer cells. Analysis of the cell-surface proteome indicated a strong link between MASTL and the regulation of TGF-β receptor II (TGFBR2), a key modulator of TGF-β signaling. Overexpression of wild-type and kinase-dead MASTL in normal mammary epithelial cells elevated TGFBR2 levels. Conversely, MASTL depletion in breast cancer cells attenuated TGFBR2 levels and downstream signaling through SMAD3 and AKT pathways. Taken together, these results indicate that MASTL supports stemness regulators in pluripotent and cancerous stem cells.Peer reviewe