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Screening for Genes That Regulate the Differentiation of Human Megakaryocytic Lineage Cells

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Screening for Genes That Regulate the Differentiation of Human Megakaryocytic Lineage Cells Screening for genes that regulate the differentiation of human megakaryocytic lineage cells Fangfang Zhua,b,1, Mingye Fengc, Rahul Sinhaa,b, Jun Seitaa,b,2, Yasuo Moria,b,3, and Irving L. Weissmana,b,d,e,1 aInstitute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305; bLudwig Center for Cancer Stem Cell Research and Medicine, Stanford University School of Medicine, Stanford, CA 94305; cDepartment of Immuno-Oncology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010; dDepartment of Pathology, Stanford University School of Medicine, Stanford, CA 94305; and eDepartment of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305 Contributed by Irving L. Weissman, July 27, 2018 (sent for review April 16, 2018; reviewed by Hongkui Deng and Lishan Su) Different combinations of transcription factors (TFs) function at genes, such as KLF1 (erythroid) and GABPA, FLI1, and RUNX1 each stage of hematopoiesis, leading to distinct expression patterns (megakaryocytic) (20, 21). They coordinate the prevention of pro- of lineage-specific genes. The identification of such regulators and genitor maintenance and the activation of downstream lineage- their functions in hematopoiesis remain largely unresolved. In this specific genes and the combination of some of those genes have study, we utilized screening approaches to study the transcriptional recently been reported to either convert human and murine fibro- regulators of megakaryocyte progenitor (MkP) generation, a key blasts to MkPs (22, 23) or promote megakaryocyte generation from step before platelet production. Promising candidate genes were human pluripotent stem cell (hPSC) lines (13). Although the results generated from a microarray platform gene expression commons are encouraging, identification of megakaryocyte-unique master and individually manipulated in human hematopoietic stem and regulators, especially those involved in MEP differentiation to MkP, progenitor cells (HSPCs). Deletion of some of the candidate genes will enable avenues for MkP and platelet generation and for (the hit genes) by CRISPR/Cas9 led to decreased MkP generation mechanistic study of their regulation. during HSPC differentiation, while more MkPs were produced when The CRISPR/Cas9 adaptive immune system, originally found some hit genes were overexpressed in HSPCs. We then demonstrated in bacteria to confer resistance to foreign genetic elements, was that overexpression of these genes can increase the frequency of demonstrated to mediate efficient and precise cleavage at en- mature megakaryocytic colonies by functional colony forming unit- dogenous genomic loci in human cells (24, 25). Single-guide CELL BIOLOGY megakaryocyte (CFU-Mk) assay and the release of platelets after in RNA (sgRNA) can be synthesized to target the specific geno- vitro maturation. Finally, we showed that the histone deacetylase mic loci, and Cas9 can induce DNA double-strand breaks inhibitors could also increase MkP differentiation, possibly by regu- (DSBs), which may generate insertion/deletion mutations and lating some of the newly identified TFs. Therefore, identification of such regulators will advance the understanding of basic mechanisms result in a loss-of-function allele. Therefore, using an sgRNA library to modify specific genomic loci by CRISPR/Cas9 suggests of HSPC differentiation and conceivably enable the generation and – maturation of megakaryocytes and platelets in vitro. a way to interrogate gene function on a large scale (26 28). megakaryocyte progenitor | transcription factors | screening | gene editing Significance erived from megakaryocytes, platelets play a major role in Megakaryocyte progenitors (MkPs), derived from hematopoietic Dhemostasis, thrombosis, inflammation, and vascular biology, stem cells (HSCs), play major roles in hemostasis, thrombosis, in- and platelet transfusions are frequently utilized to prevent throm- flammation, and vascular biology through generating platelets. bocytopenia, which can result from cancer therapy, trauma, sepsis, However, the regulatory factors involved in MkP differentiation as well as blood disorders (1). Unfortunately, the supply of these from HSCs are largely unknown. Here, we utilized a unique ge- short-lived platelets currently come with the high cost of main- nomic approach, including the microarray gene expression com- taining quality donors, the extensive testing protocols to prevent mons platform, CRISPR/Cas9-mediated gene deletion, lentivirus- contamination or recipient infection, and the generation of allo- mediated gene overexpression, as well as multicolor flow antibodies to the platelets which limit the donor pool. Another cytometry and functional assays, and identified 10 genes that are promising strategy is to transplant ex vivo-generated megakaryo- highly expressed in MkPs and required for and can promote MkP cytes (2–5), or megakaryocyte progenitor cells (MkPs), the direct generation from HSCs. In addition, we found inhibition of histone precursor for megakaryocyte, which have proliferation capacity and deacetylase activity increased MkP differentiation. Our results will engraftment potential and may therefore provide a better clinical not only shed light on the regulations of MkPs, but also facilitate alternative to standard transfusions,orasatargetforactivityin- efficient generation of MkPs and platelets for clinical applications. ducers (6, 7). Although MkPs were identified many years ago (7), the regulatory factors involved in their differentiation from hema- Author contributions: F.Z. and I.L.W. designed research; F.Z., M.F., R.S., and Y.M. per- formed research; J.S. contributed new reagents/analytic tools; F.Z. analyzed data; F.Z. topoietic stem and progenitor cells (HSPCs) are largely unknown. wrote the paper; and I.L.W. revised the paper. During hematopoiesis, transcription factors (TFs) control in- Reviewers: H.D., Peking University; and L.S., University of North Carolina at Chapel Hill. duction and maintenance of the expression of lineage-specific The authors declare no conflict of interest. genes and suppression of competing gene expression of other lineages (8–14). MkPs are originally derived from hematopoietic Published under the PNAS license. 1 stem cells (HSCs) through a well-documented stepwise differ- To whom correspondence may be addressed. Email: [email protected] or irv@stanford. edu. entiation (15, 16). To date, only a few TFs have been reported to 2Present addresses: Medical Sciences Innovation Hub Program, RIKEN, Nihonbashi, 103- be involved in this process, including AML1, FLI1, GABPA, 0027 Tokyo, Japan; and Center for Integrative Medical Sciences, RIKEN, Yokohama, 230- GATA1, RUNX1, NFE2, SCL, GATA2, MYB, and LMO2 (17– 0045 Kanagawa, Japan. 19). The bipotent megakaryocyte-erythroid progenitors (MEPs) 3Present address: Department of Medicine and Biosystemic Science, Kyushu University can directly give rise to MkPs and erythroid progenitors (EPs), Graduate School of Medical Sciences, 812-8582 Fukuoka City, Japan. which further develop into megakaryocytes and erythrocytes, re- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. spectively (20). MkPs and EPs share some TFs, including GATA1, 1073/pnas.1805434115/-/DCSupplemental. FOG1, SCL, and GFI1b, but also have several lineage-specific www.pnas.org/cgi/doi/10.1073/pnas.1805434115 PNAS Latest Articles | 1of9 Downloaded by guest on October 1, 2021 + In this study, we report a strategy to identify regulators for MkP (EP/erythrocyte marker) cells, and CD71 (erythrocyte marker) generations by genetic manipulation. Sixty candidate genes were cells (15, 35) to determine the differentiation efficiency. Flow first generated from the gene expression commons (GEXC) cytometry analysis of cell mixtures after 7–10 d of differentiation microarray platform based on their high expression level in MkPs showed the five cytokines TPO, SCF, FLT3, IL3, and IL6 in andlowinMEPsandEPs.ThenCRISPR/Cas9-mediatedgene serum-free expansion medium II (SFEMII) can lead to the highest + + + knockout as a negative screen and lentiviral-mediated gene over- percentage of CD34 CD41 MkP cells and CD41 megakaryo- expression as a positive way were utilized to determine gene func- cyte cells. Under the five-cytokine mixture culture condition, + + tions on the modulation of generation of MkPs from HSPCs. By CD34 CD41 cells represented ∼10% of the cell population after gene expression analysis, multicolor flow cytometry, colony forming in vitro differentiation (SI Appendix,Fig.S1B), and the cell can unit-megakaryocyte (CFU-Mk) functional assay, 10 regulatory genes expand 50- to 100-fold. Therefore, the five-cytokine mixture was (the hit genes) from 60 candidates were identified. Furthermore, we used for the differentiation from HSPCs into MkPs, and then showed the hit genes could promote the generation of megakaryo- TPO, SCF, and IL6 are used for megakaryocyte maturation and cytes as well as platelets. Finally, we found that inhibition of histone platelet generation in an additional 1- to 2-wk culture (Fig. 2A). deacetylase (HDAC) activity could also promote MkP differentia- The additional culture will expand cells by an additional 50- to tion, possibly by regulating some of the hit genes. 100-fold and finally each megakaryocyte can give rise to thousands of platelets. Results We then used lentiviral-mediated transduction to deliver gene Identification of Candidate TFs for MkPs
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