Direct generation of human naive induced pluripotent stem cells from somatic cells in microfluidics

Abstract

Induced pluripotent stem cells (iPSCs) are generated via the expression of the transcription factors OCT4 (also known as POU5F1), SOX2, KLF4 and cMYC (OSKM) in somatic cells. In contrast to murine naive iPSCs, conventional human iPSCs are in a more developmentally advanced state called primed pluripotency. Here, we report that human naive iPSCs (niPSCs) can be generated directly from fewer than 1,000 primary human somatic cells, without requiring stable genetic manipulation, via the delivery of modified messenger RNAs using microfluidics. Expression of the OSKM factors in combination with NANOG for 12 days generates niPSCs that are free of transgenes, karyotypically normal and display transcriptional, epigenetic and metabolic features indicative of the naive state. Importantly, niPSCs efficiently differentiate into all three germ layers. While niPSCs can be generated at low frequency under conventional conditions, our microfluidics approach enables the robust and cost-effective production of patient-specific niPSCs for regenerative medicine applications, including disease modelling and drug screening.

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Fig. 1: Devising a strategy for the transgene-free generation of naive-like iPSCs.
Fig. 2: Optimized generation and stabilization of the naive phenotype.
Fig. 3: A naive-state transcriptome is rapidly acquired during niPSC generation.
Fig. 4: Global reduction in repressive epigenetic modifications in niPSCs.
Fig. 5: Analysis of imprinted and X-linked genes status.
Fig. 6: Enhanced mitochondrial activity in niPSCs.
Fig. 7: Characterization of the differentiation potential of niPSCs.
Fig. 8: Comparison of naive iPSC generation under microfluidic conditions and CCC.

Data availability

RNA-seq and RRBS data of this study have been deposited in the Sequence Read Archive (SRA) under BioProject number PRJNA381757 and GEO under accession code GSE110377. Accession numbers of other published datasets are reported in each figure plotting RNA-seq data and in Supplementary Table 2. Source data of all repeats of all experiments are provided in Supplementary Table 5. For figure panels showing representative images of morphologies or immunostainings, images from additional independent repeats are available at Figshare (https://doi.org/10.6084/m9.figshare.c.4250195). All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

The authors thank M. Montagner and S. Dupont for critical reading of the manuscript, and the Martello Laboratory for discussions and suggestions. Thanks are also given to the following: the Smith Laboratory for sharing reset H9 cells and plasmids; Miltenyi Biotec for providing mmRNAs; A. Rosa and R. De Santis for their help with the neuronal differentiation of niPSCs; and A. Manfredi, TIGEM NGS and the Bioinformatics Core for their technical support on library generation and data processing. The authors are indebted to P. Brun for providing primary skin fibroblasts. G.M.’s Laboratory is supported by grants from the Giovanni Armenise–Harvard Foundation, the Telethon Foundation (TCP13013) and an ERC Starting Grant (MetEpiStem). D.C.’s Laboratory is supported by grants from the Giovanni Armenise–Harvard Foundation, the Telethon Foundation, the Rita Levi Montalcini programme from MIUR and an ERC Starting Grant (CellKarma). C.R.’s Laboratory is supported by the Italian Association for Cancer Research (IG17185). M.J.Z. is supported by a BMBF eMed grant (01ZX1504) and the Max Planck Society. N.E.’s Laboratory is supported by grants from the University of Padova (TRANSAC and PRAT), the CaRiPaRo Foundation, the Telethon Foundation (GGP15275), an Oak Foundation Award (W1095/OCAY-14-19) and the NIHR GOSH BRC. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.

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Authors

Contributions

S.G. and M.P. performed reprogramming, isolation, characterization and differentiation of niPSCs. I.Z. helped in establishing conditions for the expansion of naive PSCs and characterizing niPSCs. O.G. helped with the microfluidic experiments. P.M. and C.R. performed the bioinformatic analyses. D.C., M.J.Z. and G.M. designed the RRBS experiment, and M.M. and M.J.Z. analysed the RRBS data. S.G., M.P. and G.M. designed the experiments. N.E. and G.M. supervised the study and wrote the manuscript. S.G. and M.P. edited the manuscript.

Corresponding authors

Correspondence to Nicola Elvassore or Graziano Martello.

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Competing interests

S.G., M.P., N.E and G.M. are co-inventors on a patent filing describing the generation of human naive iPSCs from somatic cells. All other authors declare no competing interests.

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Integrated supplementary information

Supplementary Figure 1 Reprogramming of fibroblasts with different media.

(a) Microfluidic chip design for reprogramming. Each chip is built on common microscope glass slides and can be placed in a standard Petri dish with a phosphate buffer bath to ensure a sterile humidified environment. Fresh medium is dispensed using a standard micropipette through the inlet of the microfluidic channel. The exhausted medium is then collected from the outlet on the other end of the channel. Schematics adapted from Luni et al8. (b) DPPA5, a gene associated with naïve pluripotent cells, is expressed during the later stages of conventional primed reprogramming in microfluidics. Expression of DPPA5 is calculated relative to naïve Reset H9 cells in 2iLGo10, in red. GAPDH served as a loading control. Bars indicate mean±s.e.m. of n=3 independent experiments, shown as dots. Significant induction relative to D-1 was observed from D12 onwards. Unpaired two-tailed Mann-Whitney U test (c) Fibroblasts were exposed to 2iL from the beginning of the reprogramming protocol. In parallel we performed a conventional reprogramming in PRM as a control. We observed that by day 6 in PRM fibroblasts executed the Mesenchymal to Epithelial transition (MET)12 (top left, see also Fig. 1e). In contrast, fibroblasts exposed to 2iL underwent senescence by day 6 (bottom left and Supplementary Figure 1d) and failed to execute MET. By co-transfection of a nuclear localized GFP (nGFP) mmRNA we also observed that in PRM cells efficiently expressed nGFP, while 2iL had a negative impact on transfection efficiency on primary fibroblasts (left). At day 12 we did not observe formation of colonies in the constant presence of 2iL, whereas cells reprogrammed in parallel in PRM generated several colonies (right panels) that could be expanded as primed iPSCs, giving rise to a stable line called HPD00. Representative pictures of 2 biologically independent experiments. (d) Fibroblasts cultured in 2iL under CCC for 5 days show limited proliferation, pronounced cell flattening and spreading compared to control in conventional expansion medium containing FBS (left). EdU assay and nuclear staining confirm scarce proliferation in 2iL with enlarged and flattened nuclei (center). Reduced proliferation and senescence status after 5 days in 2iL are also indicated by decreased expression of cyclin CCNA2 and increased interleukin IL6, respectively. Expression relative to the highest value was calculated. Mean of 2 biologically independent experiments, shown as dots. GAPDH served as loading control. (e) Fibroblasts were daily transfected for 6 days using OSKMN and nGFP to monitor the efficiency of transfection and were exposed to the media and inhibitors indicated above each panel. Only fibroblasts transfected in PRM alone or with the addition of CH were efficiently transfected. Representative pictures of 2 biologically independent experiments. (f) We reinvestigated the effect of addition of LIN28 mmRNA to the OSKMN cocktail in 2iLGo+KSR+Ri in hypoxia (Fig. 1g,h) and observed a marked reduction in efficiency, in line with the role of LIN28 as a factor promoting primed pluripotency (Zhang et al., 2016). Bars indicate means of technical replicates (dots, n=45 and 9 pooled from 5 and 2 independent experiments for -LIN28 and +LIN28 conditions, respectively) shown in different shades of colours. (g) Immunofluorescence on freshly BJ-derived niPSCs (D15) using different naïve supporting media from day 6 of the reprogramming protocol. Tiling of entire microfluidic channel is shown. Fibronectin (Fn) is used as initial coating. Representative pictures of 2 biologically independent experiments. (h) Left, immunofluorescence on freshly BJ-derived niPSCs (D15) using different initial coatings: Matrigel (Mt), Laminin (Ln) or Fibronectin (Fn, shown in g). Tiling of entire microfluidic channel is shown. Right, quantification of colonies for each condition. Primary colonies were quantified at day 15 either by their compact morphology (green dots) or by the expression of KLF17 and POU5F1/OCT4 (red dots) after immunostaining. Bars indicate means of technical replicates (dots, n=8, 10 and 10 for Fn, Mt and Ln, respectively) pooled from 2 independent experiments shown in different shades of colours. Scale bars (d) 50 µm, (c, e) 200 µm (c, D12 PRM) 100 µm. Images from additional repeats for panels c, e, g and h have been deposited in Figshare under accession number 10.6084/m9.figshare.7151915. See Supplementary Table 5 for source data.

Supplementary Figure 2 Immunofluorescence staining of freshly-derived naïve-like colonies.

(a) Top view of entire microfluidic culture channels at the end of reprogramming (top) and high magnification photos (bottom) acquired on an automated epifluorescence microscope or a confocal microscope, respectively. Naïve-like colonies derived from BJ fibroblasts at day 15 (12 days of reprogramming followed by 3 days without transfection to allow clearance from mmRNA-derived proteins) express various naïve-associated markers (TFCP2L1, KLF17, DNMT3L, DPPA3, TFE3, KLF4) together with shared pluripotency markers (NANOG, POU5F1) and did not show significant levels of a primed-related marker (SSEA4). See also Supplementary Figure 1a for a description of the microfluidics culture system. n=2 independent biological experiments. (b) Top view of microfluidic culture channels at the end of reprogramming and relative magnifications using HFF-1 as an alternative reprogramming sample. Colonies show expression of naïve markers comparable to reprogrammed BJ samples. n=2 independent experiments. (c) niPSCs derived from female WI-38 lung fibroblasts at passage 3 show robust expression of naïve markers. (d) niPSCs (passage 8) derived from primary fibroblasts (passage 5) isolated from an 80 years old female donor. HPD09 niPSCs express several naïve markers, with no detectable expression of primed or differentiation markers. High magnification images from a second independent experiment for panels a and b have been deposited in Figshare under DOI 10.6084/m9.figshare.7151933. Scale bars (a-b, d) 50 µm, (c) 20 µm.

Supplementary Figure 3 niPSCs cultures and DNA characterization.

(a) Bright-field cultures of different niPSCs lines. Numerous and homogenous dome-shaped colonies are prolonged culture, indicating a stable phenotype. Images of additional independent repeats have been shared on Figshare as DOI 10.6084/m9.figshare.7151939. (b) Clonal assay of different niPSCs lines replated as single cells (2000 cells per well of a 12 well plate) in presence of murine feeders. The number of AP-positive colonies is reported. Similar results were obtained in n=3 biologically independent experiment. Images from two additional experiment have been deposited in Figshare under DOI 10.6084/m9.figshare.7151801. (c) DNA content assessed by propidium iodide and cytofluorimetric analysis. Only HPD01 (p10) and HPD07 (p23) showed a preponderant tetraploid population after several passages in CCC and RSeT. Six passages after sorting (p16), HPD01 showed a diploid-like profile ad was used thereafter for other characterizations. DNA content is routinely measured during expansion of niPSCs. Representative samples are presented. Bottom panels show the gating strategy: the gate on the FSC-A/SSC-A plot (left) eliminated cell debris, while the gate on the plot FSC-A/FSC-W (right) eliminated cell doublets. See also panel d. (d) Q-banding of niPSC lines showing normal karyotypes. Similar results were obtained in 12/12 metaphases for HPD02, 14/14 metaphases for HPD03, 11/11 metaphases for HPD04 and 12/12 metaphases for HPD06. Scale bars (a) 200 µm, (b) 1 mm.

Supplementary Figure 4 Unsupervised clustering of RNA-Seq data.

(a) Heatmap of all differentially expressed genes shows three main clusters that distinguish somatic cells, primed PSCs and naïve PSCs. The transcriptional profile of niPSCs is comparable to other human naïve PSCs. Primed PSCs obtained from BJ fibroblasts by expression of OSKMN mmRNAs in microfluidics (Fig. 1e) called HPD00 display a transcriptome similar to other primed PSCs. (b) Heatmap of unsupervised hierarchical clustering based on pluripotency associated markers of data from Kilens et al. Human iPSCs were generated in 4 different media and profiled by DGE-Seq. Expression values in UMI Per Million (UPM), obtained from Supplemental Data 1 of Kilens et al., were log2 transformed and row centred. Naïve HNES1 cells in t2iLGö and primed H1 and H9 hESCs in mTeSR1 serve as controls. Note that iPSCs generated in RSeT either cluster with other naïve PSCs cultured in t2iLGö, or are in separate clusters with an intermediate phenotype. (c) Principal components analysis (PCA) of RNA-sequencing samples from this study together with DGE-Sequencing samples from Kilens et al. Naïve HNES1 cells in t2iLGö and primed H1 and H9 hESCs in mTeSR1 (empty circles) serve as controls. To allow comparison between data generated with two different techniques, expression values were z-scored (see Methods). In the PCA only Differentially Expressed Genes used in Fig. 3c were analysed for consistency. Naïve iPSCs generated in this study cluster together with naïve PSCs expanded in t2iLGöY by Kilens and colleagues. Note also that iPSCs generated in RSeT (pink) show either a naïve or intermediate phenotype. (d) Heatmap showing expression of various genes monitored at different days of reprogramming towards primed or naïve pluripotency. Mean-normalised values are expressed in log2 scale. See also Supplementary Figure 8 for a comparison of different media on human PSCs expansion. See Supplementary Table 5 for source data.

Supplementary Figure 5 Histone and DNA methylation in niPSCs.

(a) Histone methylation H3K9me3 distribution in H9 primed pluripotent stem cells and several niPSC lines evaluated by immunofluorescence. Nuclei of representative niPSC colonies have limited and more evenly distributed H3K9me3 signal, compared to the dense and discrete foci in primed cells, which are indicative of constitutive heterochromatin regions. The right panels show quantification by image analysis of H3K9me3 signal in randomly selected nuclei. Images from 1 additional independent repeat are shown in Supplementary Figure 2c-d and have also been deposited in Figshare under DOI 10.6084/m9.figshare.7151948. (b) Methylation profiles from RRBS analysis showing the distribution of modifications from non-methylated (0) to hyper-methylated regions (1). Both fibroblasts and primed HPD00 show a bimodal distribution, with a large fraction of hyper-methylated regions (>0.8). In contrast, niPSCs show only a small portion of hyper-methylated regions, as previously reported for human blastocysts in Smith et al., 2014 - Nature. Scale bars 10 µm. See Supplementary Table 5 for source data.

Supplementary Figure 6 niPSCs differentiation.

(a) Immunofluorescence analysis of niPSCs HPD06 and primed iPSCs HPD00 competent to differentiate in the three germ layers upon specific stimuli, n=2 biologically independent experiments conducted in different lines. (b) Immunofluorescence analysis of niPSCs-derived EB outgrows. Cell clusters expressing ectodermal, mesodermal or endodermal associated markers were detected after 22 days of spontaneous differentiation, n=3 biologically independent experiments conducted in different niPSC lines. (c) Detailed qPCR analysis of EBs after 50 days of differentiation. See also Fig. 6c. Expression relative to the highest value was calculated. Bars indicate means of 2 biologically independent experiments, shown as dots (see Supplementary Table 5 for the number and values of individual replicates). GAPDH served as loading control. Scale bars, 50 µm. See Supplementary Table 5 for source data.

Supplementary Figure 7

niPSCs terminal differentiation and reprogramming in CCC and other confined systems. (a) HPD06 niPSCs were differentiated for 15 days in hepatic-like cells expressing HNF4A and CYP3A. Images of 1 representative experiment out of 2. (b) After 22 days following a neuronal differentiation protocol, HPD06 were converted in elongated neural cells expressing typical cytoskeletal markers TUJ1 and MAP2. Images of 1 representative experiments out of 2. Scale bars 50 µm. (c) In situ immunofluorescence at day 15 after 12 days of reprogramming in microfluidic platforms with different culture channel heights: 100, 200 (standard), 500, 1000 µm. Images of 1 representative experiments out of 2. Images from an additional repeats for panels a, b and c have been deposited in Figshare under DOI 10.6084/m9.figshare.7151966. Scale bars, 50 µm.

Supplementary Figure 8

Impact of different media on the human pluripotent state. (a-b) Left: We compared primed H9 hESCs, naïve H9 cells, obtained by expression of NANOG+KLF2 in t2iLGö as described by Takashima and colleagues10, and primed H9 cells cultured in RSeT on feeders for 6 passages. H9 cells in RSeT showed significant reduction of primed markers ZIC2 and OTX2 (b), while only mild activation of naïve markers KLF4, TFCP2L1 and DPPA5, significantly lower than Reset H9 Naïve cells in 2iLGo (Unpaired two-tailed Mann-Whitney U test.) (a). This intermediate naïve phenotype is consistent with other studies, including the study from Kilens and colleagues20 (See also Supplementary Fig. 4b-c). Conversely, the niPSC line HPD01 at passage 16 in RSeT clearly show robust expression of naïve markers to levels not statistically significant from Reset H9 naïve cells, and abrogation of OTX2 and ZIC2 expression. Right: human BJ fibroblasts were reprogrammed with our optimized protocol (in microfluidic with RSeT, see Fig. 2a), primary colonies were briefly expanded out of microfluidic on feeders, divided in 3 parts and expanded in parallel for a month in RSeT, 4iLA and 2iLGo (panels a-b, right). In the 3 media we observed comparable levels of naïve markers, and reduced expression of primed markers. Such profiles are not statistically significant from those of Reset H9 naïve cells expanded in 2iLGo. These results indicate that RSeT is permissive for expansion of different pluripotent phenotypes and does not induce per se rapid and full acquisition of naïve pluripotency. Yet, in the context of reprogramming with our methods it performs equally to other naïve media for expansion of niPSCs. Bars indicate means±s.e.m. of n=3 independent experiments, shown as dots. Arrowheads indicate samples significantly different from Reset H9 Naïve cells. (c) Naïve iPCs generated in RSeT and subsequently expanded in 2iLGo were analysed by immunostaining and displayed homogenous signal for naïve markers and absence of SSEA4, OTX2 and T. See Supplementary Table 5 for source data.

Supplementary information

Supplementary Information

Supplementary Figures 1–8 and Supplementary Table legends.

Reporting Summary

Supplementary Table 1

Pluripotent stem cell lines established in this work.

Supplementary Table 2

List of sample accession from GEO/ENA databases.

Supplementary Table 3

Primary antibodies used in this work.

Supplementary Table 4

Forward and reverse primers (5’>3’) used in this study.

Supplementary Table 5

Statistic source data.

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Giulitti, S., Pellegrini, M., Zorzan, I. et al. Direct generation of human naive induced pluripotent stem cells from somatic cells in microfluidics. Nat Cell Biol 21, 275–286 (2019). https://doi.org/10.1038/s41556-018-0254-5

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