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. 2018 Jul 17;9(1):194.
doi: 10.1186/s13287-018-0926-x.

Thrombopoietin knock-in augments platelet generation from human embryonic stem cells

Leisheng Zhang  1   2 , Cuicui Liu  1   2 , Hongtao Wang  1   2 , Dan Wu  1   2 , Pei Su  1   2 , Mengge Wang  1   2 , Jiaojiao Guo  3 , Shixuan Zhao  1   2 , Shuxu Dong  1   2 , Wen Zhou  3 , Cameron Arakaki  4 , Xiaobing Zhang  1   2   4 , Jiaxi Zhou  5   6
Affiliations

Thrombopoietin knock-in augments platelet generation from human embryonic stem cells

Leisheng Zhang et al. Stem Cell Res Ther. .

Abstract

Background: Refinement of therapeutic-scale platelet production in vitro will provide a new source for transfusion in patients undergoing chemotherapy or radiotherapy. However, procedures for cost-effective and scalable platelet generation remain to be established.

Methods: In this study, we established human embryonic stem cell (hESC) lines containing knock-in of thrombopoietin (TPO) via CRISPR/Cas9-mediated genome editing. The expression and secretion of TPO was detected by western blotting and enzyme-linked immunosorbent assay. Then, we tested the potency for hematopoietic differentiation by coculturing the cells with mAGM-S3 cells and measured the generation of CD43+ and CD45+ hematopoietic progenitor cells (HPCs). The potency for megakaryocytic differentiation and platelet generation of TPO knock-in hESCs were further detected by measuring the expression of CD41a and CD42b. The morphology and function of platelets were analyzed with electronic microscopy and aggregation assay.

Results: The TPO gene was successfully inserted into the AAVS1 locus of the hESC genome and two cell lines with stable TPO expression and secretion were established. TPO knock-in exerts minimal effects on pluripotency but enhances early hematopoiesis and generation of more HPCs. More importantly, upon its knock-in, TPO augments megakaryocytic differentiation and platelet generation. In addition, the platelets derived from hESCs in vitro are functionally and morphologically comparable to those found in peripheral blood. Furthermore, TPO knock-in can partially replace the large quantities of extrinsic TPO necessary for megakaryocytic differentiation and platelet generation.

Conclusions: Our results demonstrate that autonomous production of cytokines in hESCs may become a powerful approach for cost-effective and large-scale platelet generation in translational medicine.

Keywords: Early hematopoiesis; Human embryonic stem cells; Knock-in; Platelets; Thrombopoietin.

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Conflict of interest statement

Ethics approval and consent to participate

The experiment on mice followed the internationally recognized guidelines. Meanwhile, ethical approval of animal research was signed by the Ethical Committee of the Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Science & Peking Union Medical College (approval no. KT2016011-EC-1).

Consent for publication

All authors consented to publication.

Competing interests

The authors declare that they have no financial competing interests.

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Figures

Fig. 1
Fig. 1
Establishment of TPO knock-in hESC lines. a Schematic of TPO-KI stable cell line establishment. GFP used as control (Ctr). b Phase-contrast images (top panel) and fluorescence images (bottom panel) of Ctr or TPO-KI-1/-2 H1 hESCs in mTeSR. Scale bar = 20 μm. c Identification of Ctr or TPO-KI-1/-2 H1 hESCs using agarose gel electrophoresis of amplified PCR products with designed primers listed in Additional file 1: Table S1. d qRT-PCR analysis of TPO expression in Ctr or TPO-KI-1/-2 H1 hESCs in mTeSR. All values normalized to level (=1) of mRNA in Ctr. Data shown as mean ± SEM (n = 3). **P < 0.01; ***P < 0.001. e Western blotting analysis confirms expression of TPO in Ctr or TPO-KI-1/-2 H1 hESCs in mTeSR. GAPDH used as loading control. f ELISA shows soluble TPO in supernatant of Ctr or TPO-KI-1/-2 H1 hESCs after 48 h culture in mTeSR. All values normalized to level (=1) of TPO concentration in Ctr. Data shown as mean ± SEM (n = 3). **P < 0.01; ***P < 0.001. g Immunofluorescence images of pluripotency markers (POU5F1, SOX2, NANOG) in Ctr or TPO-KI-1/-2 H1 hESCs in mTeSR. Scale bar = 40 μm. h Hematoxylin and eosin (H.E.) staining of representative structures in teratomas formed by Ctr or TPO-KI-1/-2 H1 hESCs. Scale bar = 100 μm. DAPI 4′,6-diamidino-2-phenylindole, GAPDH glyceraldehyde 3-phosphate dehydrogenase, GFP green fluorescent protein, hESC human embryonic stem cell, TPO thrombopoietin
Fig. 2
Fig. 2
TPO knock-in enhances hematopoietic differentiation of hESCs. a Schematic of hematopoietic differentiation from hESCs to HPCs by coculturing with mAGM-S3 stromal cells. b Phase-contrast images of derived HPCs from Ctr or TPO-KI-1/-2 H1 hESCs at day 10 of differentiation. Typical cobblestone-like HPCs marked with white arrow. Scale bar = 40 μm. c Representative immunofluorescence images displaying generation of CD43+ HPCs from Ctr or TPO-KI-1/-2 H1 hESCs at day 7 of differentiation. Scale bar = 40 μm. d Flow cytometry analysis showing percentage of CD43+ HPCs in Ctr or TPO-KI-1/-2 H1 hESCs at day 7. Data shown as mean ± SEM (n = 3). **P < 0.01. e Representative immunofluorescence images of CD45+ HPCs generated from Ctr or TPO-KI-1/-2 H1 hESCs at day 10. Scale bar = 40 μm. f Flow cytometry analysis showing percentage of CD45+ HPCs in Ctr or TPO-KI-1/-2 H1 hESCs at day 10. Data shown as mean ± SEM (n = 3). *P < 0.05; **P < 0.01. g, h Hematopoietic colony-forming potential of CD43+ HPCs generated from Ctr or TPO-KI-1/-2 H1 hESCs. Total CFU number (g) or individual BFU-E, CFU-E, CFU-GM, or CFU-GEMM number (h) in Ctr or TPO-KI-1/-2 H1 hESCs calculated. Data shown as mean ± SEM (n = 3). *P < 0.05; NS, not significant. bFGF basic fibroblast growth factor, BFU-E burst-forming unit erythroid, BMP4 bone morphogenetic protein 4, CFU colony-forming unit, CFU-E colony-forming unit erythrocyte, CFU-GEMM colony-forming unit granulocyte/erythroid/macrophage/megakaryocyte, CFU-GM colony-forming unit granulocyte/macrophage, Ctr control, DAPI 4′,6-diamidino-2-phenylindole, hESC human embryonic stem cell, HPC hematopoietic progenitor cell, TPO thrombopoietin, VEGF vascular endothelial growth factor
Fig. 3
Fig. 3
TPO knock-in augments megakaryocytic differentiation. a Schematic of megakaryocytic differentiation from HPCs to MKs and PLTs using mAGM-S3 stromal cell coculture. b Representative cell morphology at day 6 of megakaryocytic differentiation from Ctr or TPO-KI-1/-2 H1 hESCs. Scale bar = 40 μm. Large cells indicated by white arrows. c Distribution of sizes of megakaryocytes formed at day 6 in (a) as measured with microscopy. Data shown as mean ± SEM (n = 3). *P < 0.05. d Ploidy distribution of megakaryocytes analyzed by staining cellular DNA with propidium iodide (PI). Data shown as mean ± SEM (n = 3). *P < 0.05; **P < 0.01. e, f Flow cytometer analysis for percentage of CD41a+ (e) or CD41a+CD42b+ (f) megakaryocytes at day 3 or day 6 of MK differentiation, respectively. Data shown as mean ± SEM (n = 3). *P < 0.05; **P < 0.01. g qRT-PCR analysis of megakaryocytic-associated markers (GATA1, FLI-1, RUNX1, FOG-1, NF-E2, ITGB3) in Ctr or TPO-KI-1/-2 H1 hESCs for indicated times. ACTIN used as internal control. All values normalized to level (=1) of mRNA in Ctr at day 0 of MK differentiation. Data shown as mean ± SEM (n = 3). *P < 0.05; **P < 0.01; NS, not significant. Ctr control, HPC hematopoietic progenitor cell, IL interleukin, MK megakaryocyte, PLT platelet, TPO thrombopoietin
Fig. 4
Fig. 4
More functional platelets generated from TPO knock-in cells. a Representative morphology of proplatelets derived from megakaryocytes in Ctr or TPO-KI-1/-2 H1 hESC groups. Scale bar = 40 μm. b, c Flow cytometry analysis showing percentage of CD41a+ (b) or CD41a+CD42b+ (c) platelets in Ctr or TPO-KI-1/-2 H1 hESC groups, respectively. Data shown as mean ± SEM (n = 3). *P < 0.05; **P < 0.01. d Fold change of CD41a+CD42b+ platelets in Ctr or TPO-KI-1/-2 H1 hESC groups, respectively. Data shown as mean ± SEM (n = 3). *P < 0.05; **P < 0.01. e Comparative analyses of CD41a+CD42b+ PLTs per MKs or seeded H1 hESCs, MKs per HPCs. Data shown as mean ± SEM (n = 3). *P < 0.05; **P < 0.01. f Phase-contrast images (left panel) and fluorescence images (right panel) of PB-PLTs or cultured platelets in Ctr or TPO-KI-1/-2 H1 hESC groups. Platelets stained for β-tubulin (microtubule cytoskeleton, orange). Representative line functions for single platelet shown by distance (μm) and intensity (inset). Scale bar = 2 μm. g Distribution of maximal particle diameter of PB-PLT or cultured platelets, quantified by immunofluorescence micrographs. Sizes of platelets shown as mean ± SEM (n = 3). **P < 0.01; NS, not significant. h Immunofluorescence images of platelets bound to immobilized fibrinogen with F-actin filament in absence (left panel) or presence (right panel) of 1 U/ml thrombin. Scale bar = 5 μm. i Aggregates of a mixture of 2 × 105 Calcein-AM (red)-labeled blood or cultured platelets and 2 × 107 blood platelets. Red, β-tubulin staining of both populations. Scale bar = 5 μm. j Representative flow cytometry analysis showing percentage of P-selectin (CD62P)-positive events in gated CD41a+ platelets in indicated groups. Blood platelets (right panel) used as positive controls. Ctr control, hESC human embryonic stem cell, HPC hematopoietic progenitor cell, MK megakaryocyte, PB-PLT platelet from peripheral blood, PLT platelet, TPO thrombopoietin, PLP platelet-like particle
Fig. 5
Fig. 5
TPO knock-in partially replaces extrinsic TPO. a Representative morphology of proplatelets (white arrows) among indicated groups at day 6 (D6) during MK differentiation. Scale bar = 40 μm. b, c Flow cytometry analysis showing percentage of CD41a+ (b) or CD41a+CD42b+ (c) MKs in indicated groups at day 3 (D3) and D6, respectively. Data shown as mean ± SEM (n = 3). *P < 0.05; **P < 0.01; NS, not significant. d, e Flow cytometer analysis for percentage of CD41a+ (d) or CD41a+ CD42b+ (e) platelets in indicated groups, respectively. Data shown as mean ± SEM (n = 3). *P < 0.05; **P < 0.01; NS, not significant. f Fold change of CD41a+CD42b+ PLPs in indicated groups. Data shown as mean ± SEM (n = 3). *P < 0.05; **P < 0.01; NS, not significant. g Comparative analyses of CD41a+CD42b+ PLTs per MKs or seeded H1 hESCs, MKs per HPCs, at day 6 of megakaryocytic differentiation among four groups. Data shown as mean ± SEM (n = 3). *P < 0.05; **P < 0.01; NS, not significant. Ctr control, hESC human embryonic stem cell, HPC hematopoietic progenitor cell, MK megakaryocyte, PLT platelet, TPO thrombopoietin, PLP platelet-like particles
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