Original Article Robustness of Catalytically Dead Cas9 Activators in Human Pluripotent and Mesenchymal Stem Cells Paolo Petazzi,1,7 Raul Torres-Ruiz,1,2,7 Antonella Fidanza,3 Heleia Roca-Ho,1 Francisco Gutierrez-Agüera,1 Julio Castaño,1 Sandra Rodriguez-Perales,2 Rafael Díaz de la Guardia,1 Belén López-Millán,1 Anna Bigas,4,5 Lesley M. Forrester,3 Clara Bueno,1,5 and Pablo Menéndez1,5,6 1Josep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain; 2Molecular Cytogenetics and Genome Editing Unit, Human Cancer Genetics Program, Centro Nacional de Investigaciones Oncológicas (CNIO), 28029 Madrid, Spain; 3MRC Centre for Regenerative Medicine, University of Edinburgh, 5 Little France Drive, Edinburgh EH16 4UU, UK; 4Cancer Research Program, Institut Hospital del Mar d’Investigacions Mèdiques (IMIM), Barcelona, Spain; 5Centro de Investigación Biomédica en Red de Cáncer-CIBER-ONC, ISCIII, Barcelona, Spain; 6Instituciò Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Human pluripotent stem cells (hPSCs) and mesenchymal stro- Similarly, mesenchymal stem cells (MSCs) are self-renewing multipo- mal/stem cells (hMSCs) are clinically relevant sources for tent cells present in a wide range of tissues that are capable of differ- cellular therapies and for modeling human development and entiating into various tissues of mesodermal origin and display disease. Many stem cell-based applications rely on the ability unique immunosuppressive properties.5 MSCs represent one of the to activate several endogenous genes simultaneously to modify most promising adult stem cells being used worldwide in a wide array cell fate. However, genetic intervention of these cells remains of clinical applications involving autoimmunity, hematology- challenging. Several catalytically dead Cas9 (dCas9) proteins oncology, traumatology, and cardiology.6–8 fused to distinct activation domains can modulate gene expres- sion when directed to their regulatory regions by a specific sin- The success of human stem cell-based applications often relies on gle-guide RNA (sgRNA). In this study, we have compared the methods to precisely edit the donor/patient-specific genome and/or ability of the first-generation dCas9-VP64 activator and the to regulate gene expression. Manipulation of gene expression has second-generation systems, dCas9-SAM and dCas9-SunTag, generally involved the delivery of exogenous cDNA using expression to induce gene expression in hPSCs and hMSCs. Several stem or viral vectors. However, in recent years, we have witnessed the cell lines were tested for single and multiplexed gene activation. development of strategies to activate the expression of endogenous When the activation of several genes was compared, all three gene expression using an adapted version of the clustered regularly systems induced specific and potent gene expression in both interspaced short palindromic repeat (CRISPR)/Cas9 system. This single and multiplexed settings, but the dCas9-SAM and system was originally developed as a powerful and versatile tool for dCas9-SunTag systems resulted in the highest and most consis- genome editing relying on the endonuclease (Cas9) being directed tent level of gene expression. Simultaneous targeting of the to a specific genomic site by a single-guide RNA (sgRNA), resulting same gene with multiple sgRNAs did not result in additive in a precise break in the target DNA.9,10 Mutations in residues levels of gene expression in hPSCs nor hMSCs. We demonstrate involved in DNA catalysis have generated Cas9 proteins that lack the robustness and specificity of second-generation dCas9 acti- nuclease activity while preserving DNA binding.11,12 When fused to vators as tools to simultaneously activate several endogenous effector/activation domains such as VP64, VPR, or p65 and directed genes in clinically relevant human stem cells. to regulatory regions of a genomic locus using sgRNAs, these nuclease INTRODUCTION Human pluripotent stem cells (hPSCs), including both human em- Received 7 October 2019; accepted 23 February 2020; https://doi.org/10.1016/j.omtn.2020.02.009. bryonic stem cells (hESCs) and human induced pluripotent stem cells 7 These authors contributed equally to this work. (hiPSCs), have the unique ability to self-renew indefinitely and to Correspondence: Paolo Petazzi, PhD, Josep Carreras Leukemia Research Institute, differentiate, potentially, into all cell types of the human body.1,2 School of Medicine, University of Barcelona, Casanova 143, 08036 Barcelona, They provide an unprecedented system to interrogate early human Spain. development and to provide a potential clinically relevant cell source E-mail: ppetazzi@carrerasresearch.org Correspondence: Pablo Menéndez, PhD, Josep Carreras Leukemia Research for regenerative medicine.3,4 Moreover, hiPSCs offer a unique plat- Institute, School of Medicine, University of Barcelona, Casanova 143, 08036 Bar- form for the in vitro generation of patient-specific differentiated cells celona, Spain. for personalized therapies, disease modeling, and drug screening.3,4 E-mail: pmenendez@carrerasresearch.org 196 Molecular Therapy: Nucleic Acids Vol. 20 June 2020 ª 2020 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). www.moleculartherapy.org Figure 1. Transgenic hPSCs Expressing dCas9 Transcriptional Activators Remain Pluripotent (A) Schematic of the different dCas9 transcriptional activators used in this study, VP64, SAM, and SunTag. (B) Schematic of the generation of stable transgenic hPSC-VP64, hPSC-SAM, and hPSC-SunTag lines. (C) hESC-like morphology of representative colonies from hPSC-VP64, hPSC-SAM, and hPSC-SunTag lines. (D) RT-PCR confirming (legend continued on next page) Molecular Therapy: Nucleic Acids Vol. 20 June 2020 197 Molecular Therapy: Nucleic Acids null, catalytically dead Cas9 (dCas9) variants have been shown to the typical MSC immunophenotype (CD45 CD73+CD90+CD105+, modulate endogenous gene expression.11,13–15 Recently, Chavez Figure 2C) and differentiated equally well toward adipogenic and et al.16 compared the first-generation dCas9-VP64 activator with osteogenic lineages (Figure 2D). various second-generation dCas9 activators, including dCas9-VPR, dCas9-SAM, and dCas9-SunTag, in several human, mouse, and fly The performance of dCas-VP64, dCas-SAM, and dCas9-SunTag was cell lines and demonstrated the robustness and versatility of each sys- compared across several endogenous genes representative of ecto- tem. In this study, we set out to compare single and multiplexed gene derm (NEUROD1), endoderm (FOXA2), and mesoderm (CXCR4) activation of several endogenous genes in clinically relevant hPSCs germ layers. Three different sgRNAs were designed in the proximal and human MSCs (hMSCs) using first-generation dCas9-VP64 acti- promoter (up to 250 bp upstream of the transcription start site) for vator and the second-generation activators dCas9-SAM and dCas9- each target gene, cloned in a lentiviral vector containing a puromy- SunTag. Our data demonstrate that all dCas9 systems can induce cin-resistance cassette, and transduced into the transgenic hPSC/ specific and potent gene expression, but the second-generation sys- hMSC lines. Gene expression was then analyzed in puromycin- tems result in the highest and most consistent level of gene expression selected cells (Figure 3A). All three dCas9 activator systems demon- in these cell types. We provide guidance for laboratories wanting to strated the ability to induce robust gene expression regardless of the adopt dCas9 activator technology to modulate gene expression in locus targeted, with the second-generation systems, dCas9-SAM clinically relevant human stem cells. and dCas9-SunTag, resulting in consistently higher levels of gene expression in both hPSCs (Figure 3B) and hMSCs (Figure 3C). Over- RESULTS all, the dCas9-SAM and dCas9-SunTag systems activated gene dCas9-VP64 is a fusion between the dCas9 protein and a VP64 expression to comparable levels in hPSCs, but the dCas9-SunTag transactivating domain.17 dCas9-SAM represents a modified tended to activate gene expression to a higher level than did dCas9- dCas9-VP64 system using a sgRNA that incorporates two protein- SAM in hMSCs. These data indicate that intrinsic differences in the binding aptamers (MS2) capable of recruiting the transcriptional cellular and (epi)genetic nature of hPSCs and hMSCs may activators p65 and HSF1 (MCP-p65-HSF1) to the targeted pro- impact the performance of dCas9 activator systems. There was a moter.18 The dCas9-SunTag system is a fusion between the dCas9 slight (p > 0.05) variation in gene activation between the different protein to a tail of GCN4 peptides that can recruit up to 10 copies sgRNAs that were used to target the same locus, but no correlation of scFV-VP64, which amplifies the activation signal19 (Figure 1A). was observed between the promoter distal-proximal region targeted We set out to compare these three dCas9 activators in clinically rele- by each sgRNA and the levels of gene expression (Figures 3B and vant hPSCs and hMSCs. Two different hPSC lines were used: the 3C). Consistent with previous reports, our data show a negative cor- hESC line H9 and a hiPSC line previously generated and character- relation between the basal gene expression state and the magnitude of ized in our laboratory.20 Two distinct hMSC lines were used, one activation of a given gene in both hPSCs and hMSCs (Fig- derived from bone marrow (BM) and another from adipose tissue ure 3D).16,18,21,22 To analyze the effect of recruiting multiple dCas9 (Ad). Human PSCs and MSCs were lentivirally infected with each activator complexes to the same locus, we co-transduced the cells dCas9 system at identical multiplicity of infection (MOI) of 6 to with the three different sgRNAs directed against distinct promoter re- normalize for integration events, and transduced cells were subse- gions. Simultaneous targeting of the same gene with multiple sgRNAs quently selected using antibiotic resistance (dCas9-VP64 and did not lead to additive levels of gene expression in either hPSCs or dCas9-SAM systems) or flow cytometry (dCas9-SunTag system) hMSCs, suggesting that the most efficient individual sgRNA marks (Figure 1B). Stable expression of either dCas9 activator system did a gene activation plateau for each dCas9 activator (Figures 3B and not alter the homeostasis and potency of either hPSCs or hMSCs 3C). Of note, all three systems displayed comparable and relatively (Figures 1 and 2). Antibiotic/fluorescence-activated cell sorting few integration events in both hPSCs (between 5 and 10) and hMSCs (FACS)-selected modified hPSCs and hMSCs were maintained (between 4 and 11) (Figure 3E). for >20 and >10 passages, respectively, and retained normal morphology (Figures 1C and 2A). Each transgenic stem cell line ex- Human stem cell-based applications rely on the ability to simulta- pressed comparable levels of Cas9, and also showed appropriate neously activate several endogenous genes with the potential to modify expression of the corresponding activator components (VP64, cell fate. Dead Cas9-based transcriptional activators are especially MCP, and scFV) (Figures 1D and 2B). All transgenic hPSCs suitable to target multiple loci, requiring only the provision of one retained the expression of pluripotency-associated transcription fac- sgRNA for each gene to be targeted. We next analyzed the feasibility tors (Figure 1E) and the surface marker SSEA4 (Figure 1F), and they to multiplex gene activation in both hPSCs and hMSCs. The three formed teratomas in NSG mice comprised of tissues representing all dCas9 activator systems performed well upon multiplexing gene three germ layers (Figure 1G). Similarly, transgenic hMSCs retained activation, although the second-generation dCas9-SAM and expression of the different dCas9 system components in the different transgenic hPSC lines. (E) qRT-PCR expression of the indicated pluripotency genes in hPSC-VP64, hPSC-SAM, and hPSC-SunTag lines (n = 2). Error bars indicate SEM values. (F) Representative FACS data confirming expression of the pluripotency surface maker SSEA-4 in untransduced hPSCs, hPSC-VP64, hPSC-SAM, and hPSC-SunTag lines. Insets represent unstained cells. (G) hPSC-VP64, hPSC-SAM, and hPSC-SunTag cells similarly formed teratomas in NSG mice. Left panels show macroscopic teratomas. Right panels show cellular structures representing the three germ layers. 198 Molecular Therapy: Nucleic Acids Vol. 20 June 2020 www.moleculartherapy.org (legend on next page) Molecular Therapy: Nucleic Acids Vol. 20 June 2020 199 Molecular Therapy: Nucleic Acids dCas9-SunTag systems appeared superior to the dCa9-VP64 system at at a comparable level in hPSCs whereas the dCas9-SunTag was least in two out of the three genes targeted (Figures 4A and 4B). slightly superior in hMSCs. This indicates that intrinsic cellular and We next analyzed the top in silico-predicted off-targets of FOXA2 (epi)genetic differences between hPSCs and hMSCs may impact the sgRNA (MEXA3, FUT11, and BTBD17), NEUROD1 sgRNA performance of dCas9 activator systems. In fact, differences in chro- (CCDC88C, CLSTN1, and DUSP27), and CXCR4 sgRNA (TUSC5, matin accessibility between hESCs and hESC-derived MSCs have CHRFAM7A, and ADRA2B) and found them all consistently unal- been reported.25 We have also demonstrated that simultaneous tar- tered, demonstrating the high specificity of all dCas9 activators in geting of the same gene with multiple sgRNAs did not lead to additive both hPSCs (Figure 4C) and hMSCs (Figure 4D). Taken together, levels of gene expression in neither human PSCs nor MSCs suggesting our study demonstrates the robustness and specificity of dCas9 activa- that the most efficient individual sgRNA dictates a gene activation tors as a means to simultaneously activate multiple transcription plateau for each dCas9 activator. This is in contrast to that described factors in clinically relevant human stem cells. by Chavez et al.16 for other non-stem cell lines. This could either reflect the different nature of the cells targeted or differences in the DISCUSSION DNA delivery methods or a combination both. It is also plausible Human PSCs and MSCs are relevant cell sources that hold great that each system depends on the recruitment of different downstream promise in both basic and clinical research.4 These applications transcriptional effectors, so the availability of these within the cells largely rely on robust methods to precisely control gene expression may favor certain systems. In summary, our work reveals the robust- and to simultaneously activate multiple endogenous genes with the ness, specificity, and versatility of all dCas9 activators in single and potential to modify cell fate and/or cell function. Unfortunately, multiplexed gene activation systems in clinically relevant human long-term in vitro maintenance of stemness and precise genetic stem cells and demonstrates the superior levels of gene activation manipulation using exogenous overexpression systems has long re- by the second-generation systems. mained daunting tasks. Recently, the CRISPR/Cas9 system has arisen as a unique, powerful, and versatile tool for genome editing in a wide MATERIALS AND METHODS range of cell types, including stem cells. More recently, dCas9 variants Contact for Reagent and Resource Sharing lacking nuclease activity while preserving DNA binding have been Further information and requests for resources and reagents reported.11,12 When fused to activation domains such as VP64, should be directed to, and will be fulfilled by, Pablo Menéndez VPR, or p65 they can precisely modulate endogenous gene expression (pmenendez@carrerasresearch.org). in any given locus within the genome when directed to their regula- tory regions by a specific sgRNA, thus representing a versatile tool to hPSC and hMSC Cultures regulate locus-specific gene expression.11,13–15,23 Two hPSC lines were used: the hESC line H9 obtained from WiCell (Madison, WI, USA), and a hiPSC line previously generated in our In this study, we have compared the first-generation dCas9-VP64 laboratory from B cell progenitors.20 Both hPSCs were maintained activator with the second-generation systems dCas9-SAM and on Matrigel (BD Biosciences, San Diego, CA, USA)-coated plates in dCas9-SunTag for the first time in clinically relevant human stem hESC medium as extensively reported by our group.26–28 hESC media cells. Of note, the stable expression of each of the three dCas9 activa- were changed daily, and hPSC cultures were split weekly. BM- and tors is compatible with stem cell homeostasis and potency, which is a Ad-derived hMSC cultures were maintained in Advanced DMEM critical prerequisite for any downstream stem cell application. (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with The three activator systems show the ability to induce potent gene 10% fetal bovine serum (Sigma, St. Louis, MO, USA), L-Glutamine, expression in both single and multiplexed gene activation settings. and penicillin-streptomycin-amphotericin B (Thermo Fisher Scienti- Multiplexed gene activation in stem cells is highly desired because a fic), as previously reported by our group.29 hMSC cultures were simultaneous activation of several master transcription factors is assessed daily for changes in growth rates and morphology and split key to modulate complex transcriptional networks that dictate stem every 8–10 days. Approval for hESC/hiPSC work was obtained from cell fate. Despite initial work suggesting a general decrease in gene the ISCIII-Comisión Nacional de Garantías (26/2013). activation upon multiplexing,18,24 our data confirm the robustness of these systems as tools to activate complex transcriptional multi- dCAS9 and sgRNA Lentivectors plexed regulation.16 In line with previous studies, the second-genera- All vectors used in this study were obtained from Addgene (Cam- tion systems dCas9-SAM and dCas9-SunTag delivered higher and bridge, MA, USA), including dCAS9-VP64 (#61425), MCP-p65- more consistent levels of gene expression than did dCas9-VP64. HSF1 (#61426), dCAS9-10xGCN4 (#60903), and scFv-VP64 Interestingly, the dCas9-SAM and dCas9-SunTag systems performed (#60904). The final constructs used to generate hPSC/hMSCs stably Figure 2. Transgenic hMSCs Expressing dCas9 Transcriptional Activators Remain Multipotent (A) Phase-contrast morphology of hMSC-VP64, hMSC-SAM, and hMSC-SunTag cultures revealing normal fibroblastoid-like morphology. (B) RT-PCR confirming expression of the different dCas9 system components in transgenic hMSC cultures. (C) FACS analysis revealing bona fide MSC immunophenotype (CD45 CD73+CD90+CD105+) for hMSC-VP64, hMSC-SAM, and hMSC-SunTag cultures. (D) hMSC-VP64, hMSC-SAM, and hMSC-SunTag cultures retained osteogenic (bottom panels, alizarin red staining) and adipogenic (upper panels, oil red O staining) differentiation potential. 200 Molecular Therapy: Nucleic Acids Vol. 20 June 2020 www.moleculartherapy.org (legend on next page) Molecular Therapy: Nucleic Acids Vol. 20 June 2020 201 Molecular Therapy: Nucleic Acids Figure 4. Multiplexed Activation of Endogenous Genes and Off-Target Analyses in Both hPSCs and hMSCs Both hPSCs (A) and hMSCs (B) were simultaneously transduced with three sgRNAs, one for each gene. Gene expression is shown as fold change relative to non- transduced (NT) cells. (C and D) Analysis of the top in silico-predicted off-targets of FOXA2 sgRNA (MEXA3, FUT11, and BTBD17), NEUROD1 sgRNA (CCDC88C, CLSTN1, and DUSP27), and CXCR4 sgRNA (TUSC5, CHRFAM7A, and ADRA2B) in hPSCs (C) and hMSCs (D). Error bars indicate SEM values. Statistical significance was determined by a Student’s t test (two-tailed). *p < 0.05, **p < 0.01. n = 4 independent experiments. Virus Production and Transduction of hPSCs/hMSCs A second-generation lentiviral production sys- tem was used to produce viral particles in HEK293T cells. The psPAX2 packaging plasmid, pMD2.G envelope, and the lentiviral transfer vector were co-transfected using polye- thyleneimine (PEI) (Polysciences, Warrington, PA, USA) as previously detailed.30 Virus-con- taining supernatants were harvested 48–72 h post-transfection, concentrated by ultracentri- fugation and titered in 293T cells. For transduc- tion, hPSCs/hMSCs were split 48 h before exposure to viral supernatants (MOI of 6). In- fected cells were expanded in the presence of blasticidin (3 mg/mL for VP64) and blasticidin plus hygromycin (3 and 60 mg/mL for SAM). SunTag-infected cells (transduction efficiency, 1%–13%) were FACS sorted (>95% purity) us- ing GFP and blue fluorescence protein (BFP) re- expressing the different transcriptional activators are as follows: VP64 porters, thus generating hPSCs/hMSCs stably expressing the different (dCAS9-VP64), SunTag (dCAS9-10xGCN4 + scFv-VP64), and SAM transcriptional activators (Figure 1B). Puromycin (0.3 mg/mL) was (dCAS9-VP64 + MCP-p65-HSF1) (Figure 1A). For sgRNA delivery, added to Cas9 activator-expressing hPSCs/hMSCs to select for guide both the lentiGuide-Puro (#52963) or sgRNA(MS2)_puro (#73795) RNA (gRNA) integration. backbone was Golden Gate cloned with all the guide variants accord- ing to the established protocol.18 NEUROD1 and CXCR4 sgRNA se- Quantitative Real-Time PCR quences were taken from Chavez et al.16 The best scored FOXA2 Total RNA was extracted with a Maxwell RSC simplyRNA cells kit sgRNA sequences were defined with the CRISPRa/i sgRNA designer (Promega, Madison, WI, USA) and subsequently incubated with tool from the Broad Institute. The individual sgRNAs targeting Turbo DNase (Thermo Fisher Scientific) to remove potential genomic FOXA2 at bp 42, NEUROD1 at bp 221, and CXCR4 at bp 162 contamination. Reverse transcription was performed with 500 ng of were used in multiplexing gene activation experiments. RNA using SuperScript III and random hexamer primers (Thermo Figure 3. Direct Comparison of the Three dCas9 Transcriptional Activators in Both hPSCs and hMSCs (A) Schematic workflow for the dCas9-mediated transcriptional activation. (B and C) Gene expression analysis by qRT-PCR for FOXA2 (endoderm), NEUROD1 (ectoderm), and CXCR4 (mesoderm) in hPSCs (n = 4 independent experiments using hESC and iPSC lines) (B) and hMSCs (n = 4 independent experiments with BM-MSC and Ad-MSC) (C). Three sgRNAs were tested for each gene. The genomic localization of each gRNA relative to the transcription start site (TSS) is shown. sgRNAs were used individually or pooled. Gene expression is represented as fold change relative to non-transduced (NT) cells. Error bars indicate SEM values. All statistical significance was determined by a Student’s t test (two-tailed). *p < 0.05, **p < 0.01. (D) Inverse correlation between basal gene expression and output gene activation for the three genes in both hPSCs and hMSCs. A Pearson’s test was used to determine the correlation index (r) and p value. (E) Analysis of integration events by genomic qPCR in hPSCs and hMSCs. 202 Molecular Therapy: Nucleic Acids Vol. 20 June 2020 www.moleculartherapy.org Fisher Scientific). cDNA was diluted 1:4 and 1 mL was used for each experimentation protocols were approved by the Animal Care Com- 10-mL reaction. Real-time PCR was performed with PowerUp SYBR mittee of the PRBB (Parc de Recerca Biomèdica de Barcelona). Green master mix (Thermo Fisher Scientific) in triplicate on a Bio- Rad (Hercules, CA, USA) CFX384 real-time system. All primer pairs SUPPLEMENTAL INFORMATION were designed with Primer-BLAST software and validated by gel elec- Supplemental Information can be found online at https://doi.org/10. trophoresis to amplify specific single products. A standard curve with 1016/j.omtn.2020.02.009. serial dilution of cDNA was always performed to guarantee correct amplification of primer pairs. GAPDH was used as a housekeeping AUTHOR CONTRIBUTIONS gene. Table S1 shows the sequences of all primers and gRNAs used P.P.: conceptualization, methodology, investigation, data analysis and in this study. For the off-targets analysis, the top three in silico-pre- writing. R.T.-R., A.F: conceptualization, methodology, investigation, dicted (gRNA design checker, IDT, Coralville, IA, USA) off-targets and data analysis. H.R.-H., F.G.-A., J.C., S.R.-P., R.D.d.l.G., B.L.-M., for FOXA2 42 (MEXA3, FUT11, and BTBD17), NEUROD1 221 C.B.: methodology and investigation. A.B. and L.M.F.: conceptualiza- (CCDC88C, CLSTN1, and DUSP27) and CXCR4 162 (TUSC5, tion. P.M.: conceptualization, writing and financial support. CHRFAM7A, and ADRA2B) were analyzed by real-time PCR in mul- tiplexed experiments for both hMSCs and hPSCs. CONFLICTS OF INTEREST The authors declare no competing interests. Provirus Integration Analysis Integrated lentivirus copy number analysis was performed on genomic DNA of transduced cells by using Lenti-X provirus quanti- ACKNOWLEDGMENTS We thank CERCA/Generalitat de Catalunya and Fundació Josep Car- tation kit (Takara, Japan) following the manufacturer`s guidelines. reras-Obra Social la Caixa for their institutional support. We thank Jose Luis Sardina (IJC, Barcelona) for technical assistance with the Flow Cytometry Analysis teratoma assays. Financial support for this work was obtained from The following antibodies (Becton Dickinson, San Jose, CA, USA) the Catalunya Goverment (SGR330 and PERIS 2017-2019), the Span- were used in FACS experiments: SSEA-4-V450, CD73-BV510, ish Ministry of Economy and Competitiveness (SAF2016-80481-R), CD105-FITC, CD90-allophycocyanin (APC), and CD45 APC-Cy7. the European Research Council (CoG-2014-646903), and the Funda- For staining, 200,000 cells were resuspended in 200 mL of PBS + 2% ción Leo Messi to P.M.; the Spanish Association against Cancer FBS with 1:100 antibody dilution, for 20 min at 4 C. Cells were (AECC-CI-2015) and the Health Institute Carlos III (ISCIII/FEDER, then washed twice with PBS and acquired on a FACSCanto II flow cy- PI17/01028) to C.B.; the Biotechnology and Biological Sciences tometer equipped with FACSDiva analysis software (Becton Dickin- Research Council (BBRSC) to L.M.F. and A.F.; and the Spanish Na- son, San Jose, CA, USA). tional Research and Development Plan (ISCIII/FEDER, PI17/ 02303) and the AEI/MICIU EXPLORA Project (BIO2017-91272- Adipogenic and Osteogenic In Vitro Differentiation of hMSC EXP) to S.R.-P. P.M. is an investigator of the Spanish Cell Therapy Cultures Cooperative Network (TERCEL). R.T.-R. is supported by a postdoc- hMSC differentiation was assessed by growing hMSCs in specific dif- toral fellowship from the Asociación Española Contra el Cáncer ferentiation media for 2–3 weeks according to the manufacturer’s in- (AECC). structions (Lonza, Basel, Switzerland). The detailed differentiation procedure is described elsewhere.30 Briefly, for adipogenic differenti- ation, cells were cultured in an adipogenic MSC differentiation REFERENCES 1. Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., BulletKit (Lonza), and differentiated cells were stained with oil red Marshall, V.S., and Jones, J.M. (1998). Embryonic stem cell lines derived from human O (Sigma). For osteogenic differentiation, cells were cultured in an blastocysts. Science 282, 1145–1147. osteogenic MSC differentiation BulletKit (Lonza) and differentiated 2. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and cells were stained with alizarin red-S (Sigma).29 Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibro- blasts by defined factors. Cell 131, 861–872. Teratoma Formation Assay 3. Velasco, I., Salazar, P., Giorgetti, A., Ramos-Mejía, V., Castaño, J., Romero-Moya, D., Undifferentiated hESC/hiPSC cultures at 80%–90% confluence were and Menendez, P. (2014). Concise review: generation of neurons from somatic cells of collected through enzymatic dissociation using collagenase IV, and healthy individuals and neurological patients through induced pluripotency or direct conversion. Stem Cells 32, 2811–2817. 2 million cells were re-suspended and injected with 250 mL of DMEM and 50 mL of Matrigel (BD Biosciences) subcutaneously in 4. Menendez, P., Bueno, C., and Wang, L. (2006). Human embryonic stem cells: a journey beyond cell replacement therapies. Cytotherapy 8, 530–541. the back of the NSG mice.31 Tumors generally developed within 5. Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., 6–10 weeks. When tumors reached 1-cm diameter, mice were sacri- Moorman, M.A., Simonetti, D.W., Craig, S., and Marshak, D.R. (1999). ficed and the teratomas removed and fixed overnight in paraformal- Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147. dehyde-containing solution. Teratomas were then embedded in 6. García-Castro, J., Trigueros, C., Madrenas, J., Pérez-Simón, J.A., Rodriguez, R., and paraffin, sectioned, and stained for hematoxylin and eosin to assess Menendez, P. (2008). Mesenchymal stem cells and their use as cell replacement ther- the presence of cells representing the three germ layers.31 Animal apy and disease modelling tool. J. Cell. Mol. Med. 12 (6B), 2552–2565. Molecular Therapy: Nucleic Acids Vol. 20 June 2020 203 Molecular Therapy: Nucleic Acids 7. Bernardo, M.E., and Fibbe, W.E. (2013). Mesenchymal stromal cells: sensors and and hierarchy influence the reprogramming efficiency of human B cells into induced switchers of inflammation. Cell Stem Cell 13, 392–402. pluripotent stem cells. Stem Cells 34, 581–587. 8. Asante, C.O., and Dazzi, F. (2017). Roadmap to clinical translation: insights from a 21. Chavez, A., Scheiman, J., Vora, S., Pruitt, B.W., Tuttle, M., P R Iyer, E., Lin, S., Kiani, UK regenerative medicine platform workshop on mesenchymal stromal cells. S., Guzman, C.D., Wiegand, D.J., et al. (2015). Highly efficient Cas9-mediated tran- Regen. Med. 12, 895–897. scriptional programming. Nat. Methods 12, 326–328. 9. Gasiunas, G., Barrangou, R., Horvath, P., and Siksnys, V. (2012). Cas9-crRNA ribo- 22. Fidanza, A., Lopez-Yrigoyen, M., Romanò, N., Jones, R., Taylor, A.H., and Forrester, nucleoprotein complex mediates specific DNA cleavage for adaptive immunity in L.M. (2017). An all-in-one UniSam vector system for efficient gene activation. Sci. bacteria. Proc. Natl. Acad. Sci. USA 109, E2579–E2586. Rep. 7, 6394. 10. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., and Charpentier, E. 23. Hsu, M.N., Chang, Y.H., Truong, V.A., Lai, P.L., Nguyen, T.K.N., and Hu, Y.C. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial (2019). CRISPR technologies for stem cell engineering and regenerative medicine. immunity. Science 337, 816–821. Biotechnol. Adv. 37, 107447. 11. Mali, P., Aach, J., Stranges, P.B., Esvelt, K.M., Moosburner, M., Kosuri, S., Yang, L., 24. Cheng, A.W., Wang, H., Yang, H., Shi, L., Katz, Y., Theunissen, T.W., Rangarajan, S., and Church, G.M. (2013). CAS9 transcriptional activators for target specificity Shivalila, C.S., Dadon, D.B., and Jaenisch, R. (2013). Multiplexed activation of endog- screening and paired nickases for cooperative genome engineering. Nat. enous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Biotechnol. 31, 833–838. Res. 23, 1163–1171. 12. Dominguez, A.A., Lim, W.A., and Qi, L.S. (2016). Beyond editing: repurposing 25. Gomez, N.C., Hepperla, A.J., Dumitru, R., Simon, J.M., Fang, F., and Davis, I.J. CRISPR-Cas9 for precision genome regulation and interrogation. Nat. Rev. Mol. (2016). Widespread chromatin accessibility at repetitive elements links stem cells Cell Biol. 17, 5–15. with human cancer. Cell Rep. 17, 1607–1620. 13. Gilbert, L.A., Larson, M.H., Morsut, L., Liu, Z., Brar, G.A., Torres, S.E., Stern- 26. Romero-Moya, D., Santos-Ocaña, C., Castaño, J., Garrabou, G., Rodríguez-Gómez, Ginossar, N., Brandman, O., Whitehead, E.H., Doudna, J.A., et al. (2013). CRISPR- J.A., Ruiz-Bonilla, V., Bueno, C., González-Rodríguez, P., Giorgetti, A., Perdiguero, mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, E., et al. (2017). Genetic rescue of mitochondrial and skeletal muscle impairment 442–451. in an induced pluripotent stem cells model of coenzyme Q10 deficiency. Stem Cells 35, 1687–1703. 14. Maeder, M.L., Linder, S.J., Cascio, V.M., Fu, Y., Ho, Q.H., and Joung, J.K. (2013). CRISPR RNA-guided activation of endogenous human genes. Nat. Methods 10, 27. Bueno, C., van Roon, E.H., Muñoz-López, A., Sanjuan-Pla, A., Juan, M., Navarro, A., 977–979. Stam, R.W., and Menendez, P. (2016). Immunophenotypic analysis and quantifica- tion of B-1 and B-2 B cells during human fetal hematopoietic development. 15. Qi, L.S., Larson, M.H., Gilbert, L.A., Doudna, J.A., Weissman, J.S., Arkin, A.P., and Leukemia 30, 1603–1606. Lim, W.A. (2013). Repurposing CRISPR as an RNA-guided platform for sequence- specific control of gene expression. Cell 152, 1173–1183. 28. Ramos-Mejía, V., Navarro-Montero, O., Ayllón, V., Bueno, C., Romero, T., Real, P.J., and Menendez, P. (2014). HOXA9 promotes hematopoietic commitment of human 16. Chavez, A., Tuttle, M., Pruitt, B.W., Ewen-Campen, B., Chari, R., Ter-Ovanesyan, D., embryonic stem cells. Blood 124, 3065–3075. Haque, S.J., Cecchi, R.J., Kowal, E.J.K., Buchthal, J., et al. (2016). Comparison of Cas9 activators in multiple species. Nat. Methods 13, 563–567. 29. Diaz de la Guardia, R., Lopez-Millan, B., Lavoie, J.R., Bueno, C., Castaño, J., Gómez- Casares, M., Vives, S., Palomo, L., Juan, M., Delgado, J., et al. (2017). Detailed char- 17. Perez-Pinera, P., Kocak, D.D., Vockley, C.M., Adler, A.F., Kabadi, A.M., Polstein, acterization of mesenchymal stem/stromal cells from a large cohort of AML patients L.R., Thakore, P.I., Glass, K.A., Ousterout, D.G., Leong, K.W., et al. (2013). RNA- demonstrates a definitive link to treatment outcomes. Stem Cell Reports 8, guided gene activation by CRISPR-Cas9-based transcription factors. Nat. Methods 1573–1586. 10, 973–976. 30. Prieto, C., Stam, R.W., Agraz-Doblas, A., Ballerini, P., Camos, M., Castaño, J., 18. Konermann, S., Brigham, M.D., Trevino, A.E., Joung, J., Abudayyeh, O.O., Barcena, Marschalek, R., Bursen, A., Varela, I., Bueno, C., and Menendez, P. (2016). C., Hsu, P.D., Habib, N., Gootenberg, J.S., Nishimasu, H., et al. (2015). Genome-scale Activated KRAS cooperates with MLL-AF4 to promote extramedullary engraftment transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517, and migration of cord blood CD34+ HSPC but is insufficient to initiate leukemia. 583–588. Cancer Res. 76, 2478–2489. 19. Tanenbaum, M.E., Gilbert, L.A., Qi, L.S., Weissman, J.S., and Vale, R.D. (2014). A 31. Gutierrez-Aranda, I., Ramos-Mejia, V., Bueno, C., Munoz-Lopez, M., Real, P.J., protein-tagging system for signal amplification in gene expression and fluorescence Mácia, A., Sanchez, L., Ligero, G., Garcia-Parez, J.L., and Menendez, P. (2010). imaging. Cell 159, 635–646. Human induced pluripotent stem cells develop teratoma more efficiently and faster 20. Muñoz-López, Á., van Roon, E.H., Romero-Moya, D., López-Millan, B., Stam, R.W., than human embryonic stem cells regardless the site of injection. Stem Cells 28, Colomer, D., Nakanishi, M., Bueno, C., and Menendez, P. (2016). Cellular ontogeny 1568–1570. 204 Molecular Therapy: Nucleic Acids Vol. 20 June 2020