MEIS2 regulates endothelial to hematopoietic transition of human embryonic stem cells by targeting TAL1
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Despite considerable progress in the development of methods for hematopoietic differentiation, efficient generation of transplantable hematopoietic stem cells (HSCs) and other genuine functional blood cells from human embryonic stem cells (hESCs) is still unsuccessful. Therefore, a better understanding of the molecular mechanism underlying hematopoietic differentiation of hESCs is highly demanded.
In this study, by using whole-genome gene profiling, we identified Myeloid Ectopic Viral Integration Site 2 homolog (MEIS2) as a potential regulator of hESC early hematopoietic differentiation. We deleted MEIS2 gene in hESCs using the CRISPR/CAS9 technology and induced them to hematopoietic differentiation, megakaryocytic differentiation.
In this study, we found that MEIS2 deletion impairs early hematopoietic differentiation from hESCs. Furthermore, MEIS2 deletion suppresses hemogenic endothelial specification and endothelial to hematopoietic transition (EHT), leading to the impairment of hematopoietic differentiation. Mechanistically, TAL1 acts as a downstream gene mediating the function of MEIS2 during early hematopoiesis. Interestingly, unlike MEIS1, MEIS2 deletion exerts minimal effects on megakaryocytic differentiation and platelet generation from hESCs.
Our findings advance the understanding of human hematopoietic development and may provide new insights for large-scale generation of functional blood cells for clinical applications.
KeywordsMEIS2 Hematopoiesis EHT
Endothelial to hematopoietic transition
Hemogenic endothelial cell
Hematopoietic progenitor cells
Myeloid Ectopic Viral Integration Site 2 homolog
Hematopoietic stem cell transplantation remains to be the only effective approach to treat patients with hematopoietic disorders, such as leukemia, lymphoma, and sickle cell anemia . However, the scarcity of HLA-matched HSCs severely limits the clinical applications . Thus, there is a strong need to find alternative sources to generate HSCs in vitro to meet the clinical demand. Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), represent a promising source for the generation of HSCs due to its self-renewal ability and multi-lineage differentiation potential . Since the hematopoietic differentiation capacity of hESCs was first confirmed in 2009 by Dr. James A. Thomson’s group , significant advantages have been made in the field of hematopoietic differentiation from hPSCs, including the establishment of multiple hematopoietic differentiation protocols and the generation of functional blood cells . However, derivation of bona fide HSCs with robust multi-lineage engraftment potential from hPSCs remains a major challenge . A thorough understanding of the molecular mechanism underlining hematopoietic fate decision is essential for the development of novel approaches to direct hPSCs to undergo the HSC fate.
Hematopoietic differentiation from hPSCs goes through four main stages: the emergence of mesoderm, the specification of lateral mesoderm, the generation of hemogenic endothelial cells (HEPs), and, finally, the endothelial to hematopoietic transition (EHT), with different molecular markers appearing sequentially during hematopoiesis . EHT represents a key event in hematopoietic fate commitment and is tightly regulated by extrinsic signals and intrinsic factors. It has been shown that NOTCH signaling promotes EHT, while TGFβ signaling exerts an inhibitory effect, and manipulation of these two signaling pathways markedly enhances the generation of hematopoietic cells from hPSCs [8, 9, 10]. In addition, accumulating evidence demonstrates that transcriptional factors play critical roles in hematopoietic transition from endothelium. Runx1 is considered as an essential regulator during EHT in the aorta-gonad-mesonephros (AGM) region, and enforced expression of RUNX1a enhances hematopoietic lineage commitment of hPSCs . GATA2 deletion impairs the generation of hematopoietic cells from hPSCs by suppressing EHT . TAL1 plays an important role in both HEP generation and hematopoietic transition from endothelium during hematopoiesis of hPSCs . HES1 functions downstream of NOTCH signaling and induces hematopoietic differentiation of hPSCs by refraining the endothelial commitment of HEPs . In contrast, HOXA3 and SOX7 play suppressive roles in EHT by maintaining the endothelial property of HEPs [14, 15]. Consistent with the vital role of transcriptional factors in the process of hematopoietic fate decision, an increasing number of studies have shown that blood cells can be directly induced from hPSCs or somatic cells through ectopic expression of transcription factors. For instance, ETV2/GATA2 and GATA2/TAL1 can directly convert hPSCs to blood cells with pan-myeloid or erythro-megakaryocytic potential, respectively . Hematopoietic cells with engraftment potential can be reprogrammed from human endothelial cells through expression of four transcription factors—FOSB, GFI1, RUNX1, and SPI1—in the vascular-niche microenvironment . Thus, transcription factors clearly play critical roles in hematopoietic specification. Therefore, identification of novel transcription factors in directing hematopoietic differentiation, especially at the stage of EHT, is essential for a better understanding of hematopoiesis and consequently the de novo generation of clinical-grade HSCs.
MEIS (myeloid ectopic insertion site) homeodomain transcription factors belong to the TALE (three-amino acid loop extension) superfamily and consist of three homologs MEIS1, MEIS2, and MEIS3 in human . MEIS proteins commonly function as HOX co-factors and regulate target gene transcription by directly binding to PBX proteins . Meis2 is widely expressed in mice and participates in the development of multiple organs, such as the brain, limb, lens, retina, and heart . Consistent with the function of Meis2 in mice, the patients carrying MEIS2 mutations present developmental abnormalities, including intellectual disability (ID), cleft palate, and defects in heart development [21, 22, 23]. Recent studies reported the critical role of MEIS2 in normal and malignant hematopoiesis. MEIS2 is highly expressed in AML1-ETO (AE)-positive AML and promotes leukemogenesis by directly binding to AML1-ETO and thus impairing its DNA binding ability . A recent study revealed that Meis2 is substantially upregulated in MN1 leukemic cells and mediates MN1-induced leukemic activity . Meis2-deficient mice present a small fetal liver size and display embryonic lethality between E13.5 and E14.5 , suggesting that Meis2 may influence embryonic hematopoiesis. In accordance with these observations, Meis2 overexpression promotes hematopoietic induction from mouse embryonic stem (ES) cells . However, the stage at which Meis2 regulates hematopoietic differentiation and the underlying mechanism remain undefined. In addition, whether MEIS2 has any roles in early hematopoietic differentiation in humans is still unknown. Like Meis1, mice lacking Meis2 show severe hemorrhaging , suggesting that Meis2 might also influence embryonic megakaryopoiesis just like Meis1. Consistently, enforced expression of Meis2 in mouse embryonic stem cells skews hematopoietic differentiation to megakaryocytic progenitor while suppressing erythroid differentiation . We recently reported that MEIS1 deletion severely impairs hPSC megakaryocytic differentiation and almost completely blocks platelet generation . Thus, in this study, we also explored whether MEIS2 plays similar roles in human megakaryopoiesis and thrombopoiesis. By taking advantage of genome-wide transcriptome sequencing and the CRISPR/CAS9 technology, we identified MEIS2 as a vital regulator of hESC hematopoietic differentiation. We also found that MEIS2 regulates early hematopoietic differentiation by enhancing HEP specification and EHT and does so by targeting TAL1. Together, we identify MEIS2 as a novel regulator of early human hematopoietic differentiation and provide new mechanistic insights for human hematopoiesis. Our results may facilitate the development of new strategies for large-scale generation of functional blood cells.
hESC hematopoietic differentiation in chemical defined system
The chemical defined system (CDS) was performed as previously described with some modifications to induce hESC hematopoietic differentiation. Seventy to eighty percent confluent hESCs were dissected into single cells using 1 mg/ml Accutase (Gibco) and plated on growth factors reduced (GFR) Matrigel-coated dishes at a density of 3.5 × 104 cells/well (12-well plate) in mTeSR1 medium containing 10 μM Y27632 (Calbiochem). Twenty-four hours later, hESCs were induced into stepwise differentiation. First, cells were cultured in Custom mTeSR1 medium (Stem Cell Technologies) supplemented with 50 ng/ml ActivinA (Peprotech) and 40 ng/ml BMP4 (Peprotech) for 2 days. Second, cells were incubated with Custom mTeSR1 medium supplemented with 40 ng/ml VEGF (Peprotech) and 50 ng/ml bFGF (Peprotech) for 2 days. Third, cells were incubated with Custom mTeSR1 medium supplemented with 40 ng/ml VEGF, 50 ng/ml bFGF, and 20 μM SB 431542 (STEMGENT) for 3 days. Finally, differentiated cells were dissociated and seeded into low-attachment 24-well plate and cultured for 6 days in Custom mTeSR1 medium containing 20 ng/ml SCF (Peprotech), 50 ng/ml TPO (Peprotech), 20 ng/ml IL3 (Peprotech), 1 mM glutamax (Gibco), 2% B27 (Gibco), 0.1 mM MTG (Sigma-Aldrich), 1% ITS (Gibco), 1% NAA (Gibco), and 1% penicillin/streptomycin. Fresh medium was changed every 2 days.
hESC megakaryocytic differentiation
Megakaryocytic differentiation based on mAGM-S3 co-culture system was carried out as previously described . mAGM-S3 cells were cultured to form an overgrown monolayer and treatment of 5 ng/ml mitomycin C (Sigma-Aldrich) in 37 °C for 2 h before co-culture with hESCs. hESCs were prepared as small aggregates containing about 300 cells and suspended in mTESR1 medium. The small aggregates were re-plated on mAGM-S3 cells at a density of 15–20 aggregates/well (12-well plate). On the next day, hematopoietic induction medium was used to replace mTESR1; detailed information can be found in a previous study . After 12-day culture, cobblestone cells derived from hematopoietic differentiation of hESCs were mechanically detached and seeded on mitomycin C-treated mAGM-S3 stromal cells in hematopoietic differentiation medium with the addition of Y-27632 (10 μM), TPO (50 ng/mL), SCF (20 ng/mL), IL-3 (20 ng/mL), IL-6 (10 ng/mL), and IL-11 (20 ng/mL). The medium was changed every 3 days.
Establishing MEIS2 knockout hESC lines using CRISPR/CAS9 technology
Single-guide RNA (sgRNA) targeting human MEIS2 gene was designed using the CRISPR Design Tool (http://tools.genome-engineering.org) . The sequence of corresponding oligonucleotide was cloned into lentiviral vector CRISPR-Cas9-Lenti-V2 (Addgene). Sequence-verified plasmids were denominated as Lenti-V2-MEIS2-E3G3. The genome editing efficacy of constructed plasmid was detected using surveyor in 293 T cells, and after verification, the vector was used for generating MEIS2 deletion hESC lines. To obtain MEIS2-deleted hESC lines, lentivirus containing MEIS2-E3G3 was infected into H1 hESCs, and hESCs were subsequently selected with puromycin (1 μg/ml, Sigma). After assessing the genome editing efficacy, cells were dissociated into single cells using Accutase (Gibco). Small colonies derived from individual cells were picked and expanded. Western blotting assay was performed to identify MEIS2 knockout hESC lines. hESC lines with anomalous expression of MEIS2 protein were identified by gene sequencing analysis. The sequence of primer used for surveyor assay was list as below, F: 5′-TATTTGTCGGGCTGCAGTGG-3′, R: 5′-TGTTCAAGTAGCTGGAGGCG-3′.
RNA-seq and bioinformatics analysis
RNA-seq analysis was performed by BGI Company (BGI, Shenzhen, China) as previously described . Heatmap was generated using HemI heatmap illustrator software (GPS) or R language. The RNA-seq data are available at Gene Expression Omnibus (GEO) (accession number: GSE115979).
Student’s t test was used to evaluate the differences between two groups. Differences were defined when P value was less than 0.05. Statistical analyses were performed utilizing the GraphPad Prism software. Data are presented as the mean ± SD.
MEIS2 is a potential regulator of early human hematopoietic differentiation
MEIS2 deletion impairs early hematopoietic differentiation
MEIS2 deletion suppresses HEP specification and EHT
The impairment of hematopoietic differentiation caused by MEIS2 deletion may result from at least three possibilities: (i) increased proliferation or decreased apoptosis of hematopoietic cells, (ii) suppressed generation of hematopoietic cell precursors, and (iii) impairment of EHT. To distinguish these possibilities, we first examined the effect of MEIS2 deletion on the proliferation and apoptosis of CD43+ hematopoietic cells using Ki67 incorporation and annexin V staining assays, respectively. As shown in Additional file 3: Figure S3A-B, there was no significant difference in the fraction of cycling or apoptotic cells between CD43+ hematopoietic cells derived from MEIS2+/− or MEIS2−/− hESCs and from the wild-type hESCs.
MEIS2 controls HEP specification and EHT by targeting TAL1
MEIS2 is dispensable for human megakaryopoiesis and thrombopoiesis
Elucidating the molecular mechanism governing hematopoietic lineage specification of hESCs will facilitate the generation of HSCs with long-term transplantation capacity. In the present study, we identified MEIS2 as a potential regulator of early human hematopoiesis. MEIS2 deletion inhibits hematopoietic differentiation of hESCs. Furthermore, we found that suppression of both HEP specification and EHT leads to the impairment of HPC generation. TAL1 mediates the action of MEIS2 in early human hematopoiesis. Interestingly, unlike MEIS1, MEIS2 is dispensable for megakaryopoiesis and thrombopoiesis from hESCs. Therefore, our findings not only deepen our understanding of human hematopoiesis and megakaryopoiesis but may also offer novel insights to facilitate the production of functional HSCs and other types of blood cells from hPSCs for cell-based therapies.
Meis2 knockout mice display smaller livers and severe anemia , suggesting that Meis2 may play a role in mouse embryonic hematopoiesis. However, the impact of MEIS2 deletion on human hematopoiesis remains unknown. By taking advantage of a hESC-based hematopoietic differentiation model, we found that endogenous MEIS2 expression is in parallel with hESC hematopoietic specification and is at high levels in the HEPs and CD43+ blood cells. Further functional studies demonstrated that MEIS2 deletion impairs the generation of HPCs from hESCs, confirming its critical role in early human hematopoiesis. Interestingly, MEIS2 deletion does not affect proliferation and survival of hESC-derived hematopoietic cells, in disagreement with the finding that enforced expression of Meis2 promotes mouse ESC hematopoietic differentiation by maintaining proliferation of hematopoietic progenitors . We speculate that the discrepancy between the hESC and the mouse ESC studies may be attributed to species-specific functions of MEIS2 or the difference of experimental techniques: loss-of-function analysis was used for hESCs while gain-of-function studies were conducted for mouse ESCs. Consistent with the substantial decrease of MEIS2 expression in CD45+ cells differentiated from CD43+ cells, MEIS2 deletion has little effect on the differentiation potential of hematopoietic progenitors, as shown by experiments with the CFU assay. These data suggest that MEIS2 is crucial for the induction of human hematopoietic progenitors but may be indispensable for their proliferation and differentiation into mature blood cells.
hESC hematopoietic differentiation recapitulates embryonic hematopoiesis and goes through the processes of mesoderm induction, lateral plate mesoderm specification, HEP emergence, and EHT . Our data demonstrate that MEIS2 deletion impairs hematopoietic differentiation by specifically suppressing HEP generation and EHT instead of induction and lateralization of mesoderm. As the key step of hematopoietic fate decision, EHT is regulated by transcriptional factors in a highly precise pattern during hematopoiesis. RUNX1, GATA2, TAL1, and HOXA9 have been implicated in the regulation of EHT [11, 12, 13, 33]. Combining one or more of these genes with other hematopoiesis-related genes is sufficient to directly convert hESCs or human endothelial cells into hematopoietic cells, highlighting the importance of identifying novel regulators of EHT. Therefore, it will be of great interest to explore whether MEIS2 could replace one or more genes to program hESCs or human endothelial cells into hematopoietic cells in further studies.
To understand the molecular mechanism by which MEIS2 regulates human hematopoiesis, we performed gene expression profiling of differentiated cells with or without MEIS2 deletion. As expected, MEIS2 deletion suppresses the molecular signature related to “endothelium development” and “hematopoietic transition from endothelial cells.” Combining gene expression profiling analysis with rescue experiments, we identified and confirmed TAL1 as the target gene mediating the function of MEIS2. Scl/Tal1 is widely accepted as a key factor controlling hematopoietic cell emergence . Tal1 knockout experiments reveal a vital role for Tal1 in invertebrate and mouse embryonic hematopoiesis . By using hESCs as a human developmental model, Real et al. reported TAL1 as a master regulator of human hematopoiesis . Similar to MEIS2, TAL1 regulates hematopoietic differentiation by controlling HEP specification and subsequent hematopoietic commitment. Although the role of TAL1 in human hematopoiesis is well characterized, its regulatory mechanism remains largely unknown. Our studies reveal, for the first time, that MEIS2 acts upstream of TAL1 during human hematopoiesis. The detailed mechanism underlying MEIS2 regulation of TAL1 expression during human hematopoiesis awaits future investigation.
The strong hemorrhaging phenotype in Meis2-deficient mice led us to explore the impact of MEIS2 deletion on human megakaryopoiesis and thrombopoiesis . Unexpectedly, we found that MEIS2-deleted HPCs can normally differentiate into megakaryocytes and generate platelets. The results differ from earlier reports that Meis2 promotes megakaryocyte lineage specification while suppressing erythroid progenitor differentiation in mouse ESCs . The discrepancy between the human and the mouse studies may reflect the distinct function of MEIS2 in the two species. Similar species-specific functions of other genes have been reported previously. For example, Zhang et al. showed that PAX6 is essential for neuroectoderm cell fate decision in hESCs but is not required for mouse neuroectoderm specification . Therefore, our data reiterate the distinction between humans and mice, further supporting hESCs as a unique model for deciphering the molecular basis regulating human development.
In this study, by using whole-genome gene profiling, we identified Myeloid Ectopic Viral Integration Site 2 homolog (MEIS2) as a potential regulator of hESC early hematopoietic differentiation. Furthermore, our data reveal that MEIS2 deletion inhibits hemogenic endothelial cell (HEP) specification and endothelial to hematopoietic transition (EHT). Mechanistically, TAL1 acts as the downstream gene mediating the function of MEIS2 during early hematopoiesis. Interestingly, unlike MEIS1, MEIS2 is dispensable for human megakaryopoiesis and thrombocytopoiesis, although mice lacking Meis2 show severe hemorrhaging defects.
Together, our findings advance the understanding of human hematopoietic development, and this represents important contributions to the mechanistic understanding of hPSC hematopoietic differentiation, especially during HEP specification and EHT.
The authors thank Wenying Yu, Weichao Fu, Ting Chen, and Wanzhu Yang for their technical supports.
This work was supported by the National Basic Research Program of China (2015CB964902), National Key Research and Development Program of China Stem Cell and Translational Research (2016YFA0102300, 2017YFA0103100, and 2017YFA0103102), CAMS Initiative for Innovative Medicine (2016-I2M-1-018, 2016-I2M-3-002, 2017-12 M-1-015), Chinese National Natural Science Foundation (81530008, 31671541, 31500949, 81570205), the Strategic Priority Research Program of Central South University (No. ZLXD2017004), the Hunan Province Natural Science Foundation of China (HNSF, No. 2015JJ2158), Tianjin Natural Science Foundation (16JCZDJC33100), Fundamental Research Funds for the Central Research Institutes (2016ZX310184-5), PUMC Youth Fund and Fundamental Research Funds for the Central Universities (3332015128), and PUMC Graduate Innovation Fund (2014-0710-1004, 2016-0710-09).
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information files. Meanwhile, the datasets used and analyzed during the current study are also available from the corresponding author on reasonable request.
MW contributed in the conception and design, performed the experiments and data analysis and interpretation, and wrote the manuscript. HW, YX, and WZ contributed in the conception and design. YW, YX, XL, JG, and PS performed the experiments and data analysis and interpretation. LS and JZ gave final approval of the manuscript. All authors read and approved the final manuscript.
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