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. 2016 Apr 7;7:11208.
doi: 10.1038/ncomms11208.

Large-scale Production of Megakaryocytes From Human Pluripotent Stem Cells by Chemically Defined Forward Programming

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Free PMC article

Large-scale Production of Megakaryocytes From Human Pluripotent Stem Cells by Chemically Defined Forward Programming

Thomas Moreau et al. Nat Commun. .
Free PMC article

Erratum in

Abstract

The production of megakaryocytes (MKs)--the precursors of blood platelets--from human pluripotent stem cells (hPSCs) offers exciting clinical opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chemically defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 10(5) mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufacturing practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro production of platelets for transfusion and basic research in MK and platelet biology.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. TF candidate screening for MK forward programming.
(a) TF expression in hPSCs versus cbMKs plotted as a row normalized heatmap with indications of recorded TF internal and chromatin modifiers node numbers and protein interactions among the top 20 TF candidates reported by VisANT. The nine experimentally tested TFs are highlighted in red. (b) The H9 hESC line was concurrently transduced with the 9-TFs and maintained in pluripotency medium (FGF2+Activin-A) for 2 days followed by MK medium (TPO+SCF) for a further 5 days. CD41a+ cells generated 7 days after lentiviral transduction (dot plot, mean% ±s.e.m., n=5; FL1: 530/40 nm channel) were sorted by flow cytometry and transgene expression levels quantified by RT-qPCR (n=1). (c) The percentage of CD41a+ cells was monitored by flow cytometry 7 days after transduction of the hiPSC lines #1 and #2 with the 9-TFs or 3-TFs combination. Bar graphs represent the fold increase of CD41a+ cell count relative to the 9-TFs combination (mean ±s.e.m., n=4; **P<0.01 by two-tail t-test). NT: non-transduced cells. (d) The hiPSC#1 line was transduced with all permutations of the 3-TFs and percentages of CD41a+ cells measured by flow cytometry at day 7. Bar graphs represent the fold increase of CD41a+ cell count relative to the 3-TFs combination (mean ±s.d., n=2; *P=0.06 and P<0.01 versus G+F and other combination respectively by two-tail t-test). G: GATA1, F: FLI1, T: TAL1, GFP: control vector. (e) The endogenous expression of key MK genes was monitored by RT-qPCR from CD41a flow sorted cells 7 days after transduction of the hiPSC lines #1 and #2 (mean ±s.d., n=2). (f) The hiPSC lines #1 and #2 were transduced with the 3-TFs and sorted by flow cytometry for expression of CD41a at day 7. The clonogenic potential of sorted cells was tested in methylcellulose semi-solid medium supplemented with TPO and SCF. The number of colonies per 5,000 sown cells was determined after 10 days from duplicate wells (mean±s.e.m., n=4). (g) A representative MK colony obtained from a CD41a+ cell co-expressing CD41a and CD42b as detected by immunofluorescence is shown (scale bar, 50 μm).
Figure 2
Figure 2. Generation of mature MKs by forward programming using chemically defined conditions.
(a) Schematic representation of the optimized MK-FOP protocol. Viral transduction at day 0 concurrent with embryoid body generation and mesoderm induction for 2 days was followed by a period of culture in an MK induction medium (TPO+SCF) for 8 days. Embryoid bodies showing cystic structures and actively growing cell aggregates were dissociated to single cells at day 10 and further differentiated to mature MKs (TPO+IL1β) until day 20 post transduction. (b) Time course of fopMK differentiation showing MK lineage commitment (%CD41a+ cells) and MK maturation (%CD42a+ cells) from whole culture (mean±s.e.m. from hiPSC lines #1 and #2; n=2 (day0–10); n=6 (day14–22)). Representative flow cytometry dot plots for CD41a and CD42a expression are shown below. (c) The MK fold increase at day 20 relative to the day 0 hiPSC input is shown on a logarithmic scale for the hiPSC lines #1–4 differentiated by forward programming (fopMK: mean±s.e.m.; n=14, 7, 3, 5 respectively) or directed differentiation (hiPSCs#1 and #2; ddMK: mean±s.e.m.; n=3,2 respectively). **P<0.01 and *P<0.05 by two-tail t-test. (d) Representative histograms of the expression of major platelet receptors detected by flow cytometry are shown for day 20 fopMKs (red line) and day 10 cbMKs (blue line) against isotype control (grey shade). (e) The morphology of day 20 fopMKs and day 10 cbMKs was analysed by modified Romanowsky staining on fixed cells. Arrowheads point to multinucleated cells. Scale bars, 25 μm. (f) Cell size distribution from fopMK and cbMK cultures is shown as box plots: centre lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots (n=50, 74 respectively; **P<0.01 by two-tail t-test). (g) Cell ultrastructure of fopMKs and cbMKs was visualized by transmission electron microscopy. dms, demarcation membrane system; gr, granules; mvb, multi-vesicular body; mit, mitochondria. Scale bars, 2 μm. (h) Representative confocal pictures of fopMKs immunostained for major alpha-granule proteins (thrombospondin, fibrinogen, P-selectin and Von Willebrand Factor; iPSC#3 fopMKs, day 40). Scale bars, 25 μm.
Figure 3
Figure 3. Long-term expansion of fopMKs.
(a) Schematic representation of the culture conditions for long-term MK-FOP. The combination of TPO (20 ng ml−1) and SCF (50 ng ml−1) allowed mature MK expansion for 90 days and beyond. (b) The cumulative mature MK (CD41a/CD42a double positive cells) fold increase relative to day 0 hiPSC cell input is shown for the hiPSC lines #1 (n=4) and #3 (n=5) over 90 days in culture (mean±s.e.m.). (c,d) The corresponding percentages of CD41a+ and CD42a+ cells monitored by flow cytometry from whole cultures are shown over the 90 day period (mean±s.e.m.). (e) Representative flow cytometry dot plots for CD41a and CD42a expression from the hiPSC#3 line. (f,g) Representative phase contrast picture of a day 70 hiPSC#3 fopMK long-term culture and associated Romanowsky staining of fixed cells (scale bars 50 μm). White arrowheads: clumps of actively growing small cells; red arrowheads: single big cells in suspension culture and polyploid cells identified by Romanowsky staining. (h) Cell size distribution from fopMK and LT-fopMK cultures is shown as box plots (n=50, 65 respectively). (i) Endogenous and transgenic expression levels of the 3-TFs were independently monitored by RT-qPCR throughout MK-FOP long-term cultures (mean±s.e.m. from hiPSC lines #1 and #3; n=3). The average range of expression levels in cbMKs (mean±s.e.m.; n=5) is shown as a benchmark (in red).
Figure 4
Figure 4. Transcriptional landscape of forward programmed MKs.
The undifferentiated hiPSC#1 line (n=2), day 20 fopMKs (hiPSC#1–2, n=4,2), day 25 ddMKs (hiPSC#1, n=4), day 10 cbMKs (n=4; all three groups CD42b+ sorted >95%) and day 30–69 LT-fopMKs (hiPSC#1 and #3, n=8 and 6; >80% CD42b+), were analysed for gene expression using Illumina Human HT-12 v4 BeadArrays. (a) Top five enriched gene ontology biological processes for differentially expressed (DE) genes in all MK samples compared to hiPSCs. −Log10 P values are shown as colour scale. (b) Gene set enrichment analyses for DE genes from the different MK samples (versus hiPSCs; grey circles) against a ‘MK versus other blood types’ gene expression data set (from Haematlas23). NES, normalized enrichment score; FDR, false discovery rate. (c) Gene expression correlation scatter plots on the whole-gene set using pairwise comparisons of different MK groups. R2 Pearson correlation value and differentially expressed gene numbers are indicated. The differential expression threshold (two-fold-change; FDR 5%) is shown as dotted red lines. (d) Venn diagrams recording DE genes (|Log2 fold-change| >1; FDR5%) in hiPSC-derived MKs compared with cbMKs. The number of DE genes is indicated for each intersection and the top five enriched gene ontology term biological processes from the Venn Diagram intersections are shown. (e) Hierarchical clustering using the average agglomerative method on whole-gene data set. (f) Three-dimensional plots of the principal component analysis of MK populations. The first 3 PC are shown with respective percentages of variance indicated in brackets. The two principal component analysis boxes are snapshots of a rotation along the PC3 axis with MK sample groups highlighted.
Figure 5
Figure 5. Platelet release in vitro from fopMKs throughout long-term culture.
(a) Phase contrast picture of spontaneous proplatelet-forming fopMKs (arrowheads) in suspension culture (hiPSC#3, day26; scale bar, 50 μm). (b) Proplatelet-forming fopMK on fibrinogen-coated slide immunostained for alpha-tubulin (TUBA) and P-selectin (SELP). Arrowheads indicate nascent platelet tips containing SELP-positive granules (hiPSC#4, day21; scale bar, 50 μm). (c) Transmission electron microscopy pictures of blood and in vitro-produced platelets showing typical ultrastructure. AG, alpha-granules; MVB, multi-vesicular bodies (scale bars, 1 μm). (d) Representative flow cytometry dot plots of platelet analysis. Platelets are defined within the human platelet size gate as CD41a/CD42a double positive events. (e) cbMK and fopMK from different culture time points were sown on C3H10T1/2 feeder cells for 48 h and the number of platelets released in the supernatant quantified by flow cytometry. Data represent the mean±s.e.m. of platelet number per MK sown (n=7, 4, 10, 6, 2 for cbMK and fopMK (hiPSC#1–4 pool) at day 20–90 respectively; **P<0.01 versus fopMK day 20 by two-tail t-test). (f,g) Mean platelet volume (MPV) ±s.d. and immature platelet fraction (IPF) ±s.d. of washed donor and fopMK platelets as measured on a Sysmex whole-blood analyzer (n=4/2 respectively; hiPSC#1 and #5). (h) Human platelet survival in NSG mice circulation over 24 h measured by flow cytometry following the systemic injection of 20 million washed platelets. The exponential trend of human platelet absolute count decrease over time was used for half-life calculation (mean±s.e.m.; n=5/4 for donor and fopMK (hiPSC#1 and #5) platelets respectively).
Figure 6
Figure 6. Functional assessment of fopMK in vitro platelets.
(a) Adhesion to fibrinogen of fopMK and cbMK in vitro platelets upon combined TRAP+ADP stimulation compared to blood platelets using a flow cytometry bead based assay. Percentages of adhesion on BSA or fibrinogen-coated beads are shown (mean±s.e.m., n=4, 4, 2 for blood, fopMK and cbMK respectively; **P<0.01 versus blood by two-tail t-test). (b) Representative pictures from in vitro spreading assay. Washed platelets were sown on fibrinogen-coated slides, incubated for 45 min at 37 °C and immunostained for alpha-tubulin (TUBA) and F-actin (scale bars, 10 μm). (c) Aggregation of fopMK platelets upon agonists stimulation was tested both with and compared with blood platelets using a flow cytometry-based assay: representative dot plots are shown. (d) Percentages of aggregation from Calcein-AM+ live platelets upon stimulation are shown for blood and fopMK platelets reactions (2 × 107 platelets per ml; mean±s.e.m., n=7 for each reaction group; no statistical difference versus blood at P<0.01 by two-tail t-test). (e) Associated delta-aggregation defined as ((% ADP+TRAP aggregation)—(% no agonist aggregation)) for the different reaction groups (mean ±s.e.m., n=7; P values by two-tail t-test versus blood indicated). (f) Thrombus formation in vitro under arterial shear stress. The participation of Calcein-AM live platelets spiked into human blood (at 1 × 107 per ml) is shown per 100 μm2 thrombus area. Normal or thrombocytopenic blood (>150 × 109 and <50 × 109 l−1 respectively) was used as recipient. Spiked platelets were sourced from day-8 concentrate unit (blood) or from fopMK platelets (iPSC#1 and #5; n>30 analysed thrombi per group; P values by two-tail t-test versus blood indicated). (g) Representative pictures from in vitro thrombus formation assays. Thrombi identified using bright field images are delineated and Calcein-AM platelets fluorescing in green; in vitro platelets Calcein-AM labelling is intrinsically dimmer than donor-derived platelets (Supplementary Fig. 5b). Scale bar, 50 μm. (h) Thrombus formation in vivo by laser injury of an arteriole in the cremaster muscle of NSG mice and intravital confocal microscopy. The incorporation of human Calcein-AM-labelled platelets (50 million transfused per mouse) to mouse thrombi is shown per 100 μm2 thrombus area at T_max (thrombus maximum size). Mean values±s.e.m. and P values by two-tail t-test versus donor platelets are shown (n=16/4/8 thrombi analysed for blood, fopMK#1 and #5 platelets respectively). (i) Representative snapshots of Calcein-AM+ human platelets incorporated to mouse thrombus (scale bar 10 μm).

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