Reprogramming Human Fibroblasts Into Human iPSCs

1 Materials and methods Human foreskin fibroblasts were commercially obtained (Sigma, SCC058) and reprogrammed into human-induced pluripotent stem cells using the StemMACS iPSC mRNA Reprogramming Kit, human (130-132-990) and isolated using MACS® Technology (fig. 1). Briefly, 1 × 10⁵ cells were seeded onto Laminin-coated (at 0.5 μm/cm²) wells of a 6-well plate (D-4) and expanded for three days prior to the start of mRNA transfection. Starting from day 0, mRNA transfections were performed twice a day for four days until day 4. StemMACS™ iPSC mRNA Reprogramming Kit, human Efficient reprogramming of human foreskin fibroblasts into human iPSCs Background Human-induced pluripotent stem cells (iPSCs) have the ability to differentiate into various tissues. They can be obtained from somatic patient-derived cells by reprogramming, rendering them highly potent for individualized regenerative medicine, basic and translational research, and drug screening. Reprogramming has been traditionally achieved by Sendai virus-based transduction or plasmid-based expression of the Yamanaka factors. Viral-based reprogramming methods, while widely used, require confirmed viral clearance in accordance with ISSCR and ISCT guidelines¹ and are often subject to Biosafety Level 2 regulations introducing additional regulatory and safety considerations². Reprogramming based on mRNA has the potential to overcome both of these issues, but existing solutions are expensive, have a lower safety profile, or suffer from low efficiencies³. Here, we used the StemMACS™ iPSC mRNA Reprogramming Kit, human, to reprogram commercially available human foreskin fibroblasts (HFF) into iPSCs, and characterized their pluripotency and mitochondrial DNA (mtDNA) integrity. Miltenyi Biotec products Order no. StemMACS iPSC mRNA Reprogramming Kit, human 130-132-990 Anti-TRA-1-60 MicroBeads, human 130-100-832 SSEA-4 Antibody, anti-human, VioGreen™, REAfinity™ 130-124-073 Oct3/4 Isoform A Antibody, anti-human/mouse, APC, REAfinity 130-117-821 TRA-1-60 Antibody, anti-human, PE, REAfinity 130-122-965 PAX-6 Antibody, anti-human, APC, REAfinity 130-123-328 Sox2 Antibody, anti-human/mouse, FITC, REAfinity 130-120-790 Sox17 Antibody, anti-human, Vio® B515, REAfinity 130-111-147 CD140b Antibody, anti-human, APC, REAfinity 130-121-128 CD144 (VE-Cadherin) Antibody, anti-human, FITC, REAfinity 130-123-932 J. Dobner and A. Rossi Genome Engineering and Model Development lab (GEMD), IUF-Leibniz Research Institute for Environmental Medicine, Düsseldorf, 40225, Germany Afterwards, cells were cultured in StemMACS PSC-Brew XF complemented with StemMACS PSC-Support XF, with daily medium changes except on day 6. Cultures were maintained until day 10 for iPSC isolation or day 14 for reprogramming efficiency assessment (fig. 2). Reprogrammed iPSCs were isolated on day 10 either by manual colony picking or by cell separation using MACS Technology with TRA-1-60 MicroBeads, human (130-100-832). On day 14, reprogramming efficiency was evaluated by immunofluorescent staining with antibodies against Oct4 (Cell Signaling Technologies, #2840) and TRA-1-60 (Cell Signaling Technologies, #4746). Single-cell isolated iPSCs obtained using MACS Technology were expanded for four days, seeded as single cells on day 14, further expanded, split once, and then subjected to pluripotency characterization. To that end, colony-picked and single-cell-isolated reprogrammed iPSCs were trilineagedifferentiated (StemMACS Trilineage Differentiation Kit) and functional pluripotency was assessed by flow cytometry and the qPCR-based hiPSCore assay. For flow cytometry characterization of the undifferentiated state, SSEA-4 (130-124-073), Oct3/4 (130-117-821), and TRA-1-60, (130-122-965) antibodies were used. For flow cytometry characterization of trilineage differentiation, PAX6 (130-123-328), SOX2 (130-120-790), SOX17 (130-111-147), CD140b (130-121-128), and CD144 (Miltenyi Biotec, 130-123-932) antibodies were used. Mitochondrial DNA integrity was assessed by enrichment of mtDNA in crude cell lysates and analyzed by Mitopore.4,5,6 Figure 2: Quick and efficient iPSC generation in 14 days. Timeline of reprogramming human fibroblasts into iPSCs using the StemMACS iPSC mRNA Reprogramming Kit, human. Figure 1: Schematic overview of fibroblast reprogramming into PSCs. From cell collection to reprogramming and PSC isolation, Miltenyi Biotec supports every step with reliable and time saving solutions. 2 Perform mRNA transfections twice a day StemMACS iPSC mRNA Reprogramming Kit, human Change media daily (except day 6) Isolate putative human iPSCs Assessment of reprogramming efficiency and pluripotency characterization of isolated clones Seed fibroblasts Day -3 Day 0 Day 4 Day 5 ... Day 11 ... Cell collection Easily obtain somatic cells for reprogramming. Reprogramming Efficiently reprogram somatic cells into iPS cells. reprogramming factors PSC isolation Gently isolate iPS cells from un-reprogramming cells. StemMACS PSC-Brew XF, human Results and discussion Efficient reprogramming and validation of iPSC clones By using the StemMACS iPSC mRNA Reprogramming Kit, human, foreskin fibroblasts were reprogrammed into iPSCs with high efficiency of up to 1.4% (fig. 3A). To expand iPSC clones, we followed a two-way strategy and isolated colonies either by manual picking or by manual cell separation using Anti-TRA-1-60 MicroBeads, human, followed by staining and cytometric analysis of TRA1-60 antibody (fig. 3B). After expansion, iPSCs were subjected to flow cytometry. In line with colony staining, analysis of markers of the undifferentiated state (fig. 3C) indicated successful reprogramming. Trilineage differentiation confirms functional pluripotency of iPSCs To assess functional pluripotency, iPSCs were subjected to directed trilineage differentiation for seven days (fig. 4A) and differentiation success was evaluated by flow cytometry (fig. 4B). In parallel, functional pluripotency was assessed using the qPCR-based hiPSCore assay, which revealed high confidence classification of undifferentiated iPSCs, as well as endo-, ecto-, and mesoderm-differentiated cells (fig. 4C). Together, these results demonstrate successful reprogramming, independent of subsequent isolation and expansion of reprogrammed iPSCs. mRNA-based reprogramming preserves mtDNA integrity Reprogramming induces drastic metabolic changes and has been reported to affect mtDNA stability by introducing mutations. To assess potential effects on mtDNA, we examined the integrity of reprogrammed cells using Mitopore (fig. 5). We analyzed 27 individual clones and found the mtDNA of each sample was unaltered compared to the parental HFF line, indicating that mRNA-based reprogramming does not introduce harmful alterations in the majority of cases. Figure 3: Reprogramming of human foreskin fibroblasts into induced pluripotent stem cells. (A) Staining of colonies with undifferentiated state markers Oct3/4 and TRA-1-60 revealed efficient reprogramming. Asterisks indicate iPSC colonies. (B) Reprogrammed iPSCs were either manually picked or isolated using Anti-TRA-1-60 MicroBeads, human, followed by flow cytometry analysis. (C) Resulting iPSCs displayed high purity, as assessed by staining for undifferentiated state markers SSEA-4, TRA-1-60, and Oct3/4. Efficiency = no. colonies / initial seeding density No. colonies zoom: 82 Initial seeding density: 1 x 10⁵ fibroblasts Surface area 6-well: 9.6 cm² Surface area ROI: 0.55 cm² Scaling factor: ~ 17.5 Estimated reprogramming efficiency = (82 * 17.5) / 100,000 = 1.435% 3 Oct -3/4 TRA1-60 Merge A B C FSC-A VioGreen PE APC Flow cytrometry Fibroblast iPSC Reprogramming Colony picking Figure 4: Functional characterization of reprogrammed human induced pluripotent stem cells. (A) Reprogrammed iPSCs were differentiated into endoderm (endo), ectoderm (ecto), and mesoderm (meso) using the StemMACS Trilineage Differentiation Kit. (B) Resulting primary germ layer populations were analyzed by flow cytometry. High levels of endoderm markers SOX17 (99%) and CXCR4 (100%), ectoderm markers SOX2 (100%) and PAX6 (99%), and mesoderm marker CD140b (95%) were detected. CD144 levels were low (8%), consistent with CD140b and CD144 being mutually exclusive during early mesoderm differentiation. (C) Primary germ layer populations were further analyzed using the qPCR-based hiPSCore assay and confirmed to be pluripotent, based on subtests of the undifferentiated state, endoderm, ectoderm, and mesoderm, as well as the aggregated hiPSCore. Certainty of classification ranges from 0 (lowest confidence) to 1 (highest confidence). *Clone #1 was isolated by colony picking whereas the other clones were isolated using MACS Technology. 4 Endo Ecto Meso iPSC A C Clone Undiff Endo Ecto Meso hiPSCore E11 0.83 0.88 0.98 0.92 3.61/4.00 E8 0.91 0.93 0.98 0.95 3.77/4.00 #1* 0.79 0.82 1.00 0.95 3.55/4.00 B5 0.83 0.82 1.00 0.95 3.61/4.00 E4 0.73 0.81 1.00 0.95 3.49/4.00 H3 0.83 0.82 1.00 0.95 3.59/4.00 B ecto FSC-A meso endo FITC APC References 1. Grafen, M. et al. (2017) Lab Invest. 97: 863–872. 2. Shi, S.R. et al. (1991) J. Histochem. Cytochem. 39: 741–748. 3. Corver, W.E. and ter Haar, N.T. (2011) Curr. Protoc. Cytom. Chapter 7: Unit 7.37. 4. Polioudaki, H. et al. (2015) BMC Cancer 15: 399. Conclusions This study shows that the StemMACS iPSC mRNA Reprogramming Kit, human, provides fast and efficient reprogramming of human-derived somatic cells into iPSCs. Key benefits include: • Fast reprogramming with clear timelines from fibroblast seeding to iPSC isolation. • Robust production of pluripotent iPSCs confirmed by flow cytometry and trilineage differentiation. • Preserved mitochondrial DNA integrity and compatibility with standardized workflows. Together, these results underline a practical and dependable approach for producing human iPSCs suitable for research and development. Figure 5: Analysis of mitochondrial DNA integrity of individually reprogrammed human iPSC clones. The variant landscape, based on all detected variants, demonstrates similarity between all analyzed iPSC clones and the parental fibroblast line (black rectangle). *Value is missing due to low coverage.. Manual inspection of the binary alignment map (BAM) file revealed the same variant as in all other samples. 130-140-945 Miltenyi Biotec B.V. & Co. 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