The hESC line H9 was obtained from WiCell (Madison, WI, United States), and cells were maintained on Matrigel (BD Biosciences, San Diego, CA)-coated plates and fed daily with mouse embryo fibroblast (MEF)-conditioned medium supplemented with 8 ng/mL basic fibroblast growth factor (Miltenyi, Bergisch Gladbach, Germany), as previously described by our group (Bueno et al., 2013; Romero-Moya et al., 2017). H9 cells were passaged weekly by dissociation with 0.05% trypsin (Thermo Fisher Scientific, Waltham, MA). Approval for hPSC work was obtained from the ISCIII-Comisión Nacional de Garantías (26/2013).

To provide the optimal repair template for our engineered H9 cells, we used 1 µg of the linearized iCas9.SAM plasmid (Addgene #211495). We then prepared ribonucleoprotein (RNP) complexes by mixing equimolar amounts (50 pmol) of sgRNAs (listed in Supplementary Table S1) and dCas9 protein (IDT, Coralville, IO). Finally, 2 × 10⁵ H9 cells (2.2 × 10⁷ cells/mL) were electroporated, together with the iSAM cassette and Cas9 RNPs, using the Neon system (Invitrogen, Carlsbad, CA) with the following settings: 3 consecutive pulses of 1400 V and 5 ms pulse width. Cells were immediately seeded into one well of a six-well plate coated with Matrigel and were selected with geneticin (100 μg/mL) starting 24 h later.

Genomic DNA was isolated using the QIAamp DNA Mini Kit (Qiagen, Leiden, Germany). Ten µg of DNA were digested with BglII (New England Biolabs, Ipswich, MA), separated on a 0.8% agarose gel, and transferred to a Hybond nylon membrane (RPN303B; Amersham Biosciences, Amersham, United Kingdom). Membranes were hybridized with DIG-dUTP-labeled probes. Probes were detected using a 1:5,000 dilution of AP-conjugated DIG-antibody (Roche Diagnostics, Basal, Switzerland) with CDP-Star (Sigma-Aldrich, St. Louis, MO) as a substrate for chemiluminescence. The probe was synthesized by PCR with the PCR DIG Probe Synthesis Kit (Roche Diagnostics) using plasmid DNA as a template. Primers used for probes are detailed in Supplementary Table S1.

For sgRNA delivery, the sgRNA (MS2) _puro (Addgene #73795) backbone was Golden Gate cloned with all the guide variants according to an established protocol (Konermann et al., 2014). ASCL1, NEUROD1 and CXCR4 sgRNA sequences depicted in Supplementary Table S1 were taken from (Chavez et al., 2016).

A second-generation lentiviral production system was used to produce viral particles in HEK293T cells. The psPAX2 packaging plasmid, pMD2.G envelope plasmid and the lentiviral transfer vector were co-transfected using polyethyleneimine (Polysciences, Warrington, PA), as described (Prieto et al., 2016). Virus-containing supernatants were harvested 48–72 h after transfection, concentrated by ultracentrifugation and tittered in HEK293T cells. For transduction, H9 cells were passaged 24–48 h before exposure to viral supernatants (multiplicity of infection of 10). H9-iCas9.SAM infected cells, were selected for sgRNA integration with Puromycin (0.3 μg/mL).

Total RNA was extracted with the Maxwell RSC simplyRNA Cells Kit (Promega, Madison, WI, United States). Reverse transcription was performed with 1 μg of RNA using SuperScript III and random hexamer primers (Thermo Fisher Scientific). cDNA was diluted 1:4 and 1 μL was used for each 10 μL reaction. Real-time PCR was performed with the PowerUp SYBR Green master mix (Thermo Fisher Scientific) in triplicate on the Bio-Rad CFX384 real-time platform (Hercules, CA). All primer pairs were designed with Primer-BLAST software and validated by running a standard curve with serial dilutions of cDNA to guarantee correct amplification of primer pairs. GAPDH or RPL19 were used as housekeeping genes. Supplementary Table S1 lists the sequences of all primers and sgRNAs used in this study.

Supplementary Table S2 shows the antibodies used for flow cytometry (TRA-1-60-BV510, TRA-1-81-APC, SSEA-4-V450, Nestin-APC, CD31-BV510, CD34-PECy7, CD43-FITC, CD45-APC, unconjugated SOX17 and human Serum Albumin-APC). For staining, 200,000 cells were resuspended in 200 μL of PBS+2% fetal bovine serum (FBS) and the corresponding antibodies at dilutions depicted on Supplementary Table S2, for 20 min at 4°C. Cells were then washed twice with PBS 1X and acquired on a FACSCanto II flow cytometer equipped with FACSDiva analysis software version 8.0 (Becton Dickinson, San Jose, CA, United States).

Undifferentiated H9-iCas9.SAM cultures at 80%–90% confluence were collected by enzymatic dissociation with collagenase IV, and 2 × 10⁶ cells were re-suspended and injected in a volume of 250 μL of Dulbecco’s modified Eagle’s medium (DMEM) and 50 μL of Matrigel subcutaneously in both flanks of 6- to 12-week-old NOD-Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice (The Jackson Laboratory) housed under pathogen-free conditions in the animal facility of the Barcelona Biomedical Research Park (Gutierrez-Aranda et al., 2010; Petazzi et al., 2020). Mice were euthanized when tumours reached 1 cm³ diameter. Teratomas were removed, fixed overnight in paraformaldehyde-containing solution, embedded in paraffin, sectioned, and stained with hematoxylin and eosin to assess the presence of cells deriving from the three germ layers. Animal experimentation protocols were approved by the Animal Care Committee of the Barcelona Scientific Research Park (HRH-17-0015).

Neural progenitors were differentiated using the PSC neural induction medium (NIM; Thermo Fisher Scientific, #A1647801). H9-iCas9.SAM cells were dissociated to a single cell suspension and 3 × 10⁵ cells were seeded into a Matrigel-coated 6-well plate. NIM was replaced every other day. Neural stem cells (NSCs) were passed once weekly from day 7 following manufacturer’s specifications. Differentiation assays were performed in parallel with and without supplementation of 2 μg/mL of doxycycline.

Undifferentiated H9-iCas9.SAM cells were treated with collagenase IV for 5 min and gently scraped off from the plate. The embryoid bodies (EBs) were transferred to low-attachment plates and incubated overnight in medium composed of KnockOut-DMEM (Thermo Fisher Scientific) supplemented with 20% non-heat-inactivated FBS for hESCs (Biowest, Nuaillé, France), 1X Glutamax (Thermo Fisher Scientific), 0.1 mM nonessential amino acids, and 0.1 mM β-mercaptoethanol. The medium was replenished on the next day and was supplemented with bone morphogenetic protein 4 (BMP-4) (50 ng/mL), FMS-related tyrosine kinase 3 ligand (Flt-3L) (300 ng/mL), stem cell factor (SCF) (300 ng/mL), interleukin-3 (IL-3) (10 ng/mL), interleukin-6 (IL-6) (10 ng/mL) and granulocyte-colony stimulating factor (G-CSF) (50 ng/mL), with medium changes every 3–4 days (Bueno et al., 2012; 2013; Gutierrez-Agüera et al., 2021). All growth factors and cytokines were from R&D Systems (Minneapolis, MN, United States). Differentiation assays were performed in parallel with and without supplementation of 2 μg/mL of doxycycline.

For hepatic differentiation, H9-iCas9.SAM cells were first guided into definitive endoderm (DE) in RPMI-1640 medium supplemented with 1% nonessential amino acids, 1% B27 supplement without vitamin A, 2 mM L-glutamine and 50 units/mL penicillin/streptomycin (all from Gibco-Invitrogen). Differentiation was initiated (day 0) when the cell density reached 3.2–5.2 × 10⁵ cells/cm². On day 1, the medium was changed and supplemented with 100 ng/mL Activin A, 50 ng/mL BMP-4 and 6.45 μM CHIR99021 (all from R&D Systems). From day 2 to day 5, cells were supplemented with only 100 ng/mL of Activin A. After DE differentiation, H9-iCas9.SAM cells were differentiated to hepatocyte-like cells following the Cellartis Hepatocyte Differentiation protocol (Cellartis Hepatocyte Diff Kit; Takara Bio Europe AB, Gothenburg, Sweden; Y30050). Hepatocyte Progenitor Medium was used for medium changes on days 9 and 11 of differentiation. Finally, on day 14, cells were cultured with Hepatocyte Maturation Medium Base (3A) supplemented with Hepatocyte Maturation Medium Supplement (3B) according to manufacturer’s specification. Differentiation assays were performed in parallel with and without supplementation of 2 μg/mL of doxycycline.

The potential of hPSCs in developmental biology studies and cell therapy applications hinges on their capacity to differentiate into various cell types. To explore this potential, we employed H9-iCas9.SAM cells and subjected them to directed differentiation protocols aimed at generating neural stem cells (NSCs), hematopoietic cells, definitive endoderm (DE), and hepatocytes, representing ectoderm, mesoderm, and endoderm, respectively.

NSCs, known as multipotent progenitors of neurons and glia (Shi et al., 2012), offer valuable insights into neuronal physiology and disease modeling. Using a commercial kit, we successfully cultured Nestin + NSCs within just 5 days of differentiation, with over 90% of the cells expressing Nestin (Figure 2A, left panels). Following induction with doxycycline, approximately 60% of these NSCs expressed mCherry (Figure 2A, left and middle panels), confirming the effective induction of the iSAM cassette. A specific 100-fold upregulation of the dCas9 (iSAM) cassette in the doxycycline treatment further validated the successful induction (Figure 2A, right panels). Remarkably, H9-iCas9.SAM-derived NSCs were sustained by day 13 of differentiation, exhibiting consistent levels of iSAM activation and even higher proportions (70%) of mCherry + Nestin + NSCs (Figure 2A, bottom panels).

We next induced hematopoietic differentiation from H9-iCas9.SAM cells using an EB-based differentiation protocol. During in vitro hematopoietic development, hPSCs are first specified into hematoendothelial progenitors (HEPs: CD34⁺CD31⁺), which then progress to CD45⁺ hematopoietic cells (Chadwick et al., 2003; Gutierrez-Agüera et al., 2021). CD34⁺CD31⁺ HEPs and CD45⁺ cells were consistently detectable at day 7 and day 12, respectively (Figure 2B, left panel). Following doxycycline treatment, sustained and robust expression of mCherry (Figure 2B, left and middle panels) and dCas9 (Figure 2B, right panels) was observed in both CD34⁺CD31⁺ HEPs and CD45⁺ cells, confirming successful iSAM activation upon mesodermal differentiation.

Finally, H9-iCas9-SAM cells were differentiated into hepatocytes. We first induced differentiation into SOX17⁺ DE and observed >90% SOX17⁺ cells by day 5, 55% of them expressing mCherry specifically in the doxycycline treatment (Figure 2C, left and middle panels). A massive upregulation of the dCas9 was observed in the doxycycline treatment confirming the successful activation of the iSAM (Figure 2C, right panels). Further differentiation of SOX17⁺ DE into hepatocytes rendered >98% of Albumin⁺ cells by day 14 of differentiation with a slightly lower but sustained expression of mCherry and dCas9 in ∼40% of the Albumin⁺ cells, confirming successful iSAM activation after endodermal specification (Figure 2C, bottom panels).