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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 and GABPA, FLI1, and RUNX1 (megakaryocytic) (20, 21). They each stage of hematopoiesis, leading to distinct expression patterns coordinate the prevention of progenitor maintenance and the acti- of lineage-specific genes. The identification of such regulators and vation of downstream lineage-specific genes and the combination of their functions in hematopoiesis remain largely unresolved. In this some of those genes have recently been reported to either convert study, we utilized screening approaches to study the transcriptional human and murine fibroblasts to MkPs (22, 23) or promote mega- regulators of megakaryocyte progenitor (MkP) generation, a key karyocyte generation from human pluripotent stem cell (hPSC) lines step before platelet production. Promising candidate genes were (13). Although the results are encouraging, identification of mega- generated from a microarray platform gene expression commons karyocyte-unique master regulators, especially those involved in MEP and individually manipulated in human hematopoietic stem and differentiation to MkP, will enable avenues for MkP and platelet progenitor cells (HSPCs). Deletion of some of the candidate genes generation and for mechanistic study of their regulation. (the hit genes) by CRISPR/Cas9 led to decreased MkP generation The CRISPR/Cas9 adaptive immune system, originally found during HSPC differentiation, while more MkPs were produced when in bacteria to confer resistance to foreign genetic elements, was some hit genes were overexpressed in HSPCs. We then demonstrated demonstrated to mediate efficient and precise cleavage at en- that overexpression of these genes can increase the frequency of dogenous genomic loci in human cells (24, 25). Single-guide mature megakaryocytic colonies by functional colony forming unit- RNA (sgRNA) can be synthesized to target the specific geno- megakaryocyte (CFU-Mk) assay and the release of platelets after in mic loci, and Cas9 can induce DNA double-strand breaks vitro maturation. Finally, we showed that the histone deacetylase (DSBs), which may generate insertion/deletion mutations and inhibitors could also increase MkP differentiation, possibly by regu- result in a loss-of-function allele. Therefore, using an sgRNA lating some of the newly identified TFs. Therefore, identification of library to modify specific genomic loci by CRISPR/Cas9 suggests such regulators will advance the understanding of basic mechanisms a way to interrogate gene function on a large scale (26–28). of HSPC differentiation and conceivably enable the generation and maturation of megakaryocytes and platelets in vitro. Significance megakaryocyte progenitor | transcription factors | screening | gene editing Megakaryocyte progenitors (MkPs), derived from hematopoietic erived from megakaryocytes, platelets play a major role in stem cells (HSCs), play major roles in hemostasis, thrombosis, in- Dhemostasis, thrombosis, inflammation, and vascular biology, flammation, and vascular biology through generating platelets. and platelet transfusions are frequently utilized to prevent throm- However, the regulatory factors involved in MkP differentiation bocytopenia, which can result from cancer therapy, trauma, sepsis, from HSCs are largely unknown. Here, we utilized a unique ge- as well as blood disorders (1). Unfortunately, the supply of these nomic approach, including the microarray gene expression com- short-lived platelets currently come with the high cost of main- mons platform, CRISPR/Cas9-mediated gene deletion, lentivirus- taining quality donors, the extensive testing protocols to prevent mediated gene overexpression, as well as multicolor flow contamination or recipient infection, and the generation of allo- cytometry and functional assays, and identified 10 genes that are antibodies to the platelets which limit the donor pool. Another highly expressed in MkPs and required for and can promote MkP promising strategy is to transplant ex vivo-generated megakaryo- generation from HSCs. In addition, we found inhibition of histone cytes (2–5), or megakaryocyte progenitor cells (MkPs), the direct deacetylase activity increased MkP differentiation. Our results will precursor for megakaryocyte, which have proliferation capacity and not only shed light on the regulations of MkPs, but also facilitate engraftment potential and may therefore provide a better clinical efficient generation of MkPs and platelets for clinical applications. alternative to standard transfusions, or as a target for activity in- Author contributions: F.Z. and I.L.W. designed research; F.Z., M.F., R.S., and Y.M. per- ducers (6, 7). Although MkPs were identified many years ago (7), formed research; J.S. contributed new reagents/analytic tools; F.Z. analyzed data; F.Z. the regulatory factors involved in their differentiation from hema- wrote the paper; and I.L.W. revised the paper. topoietic stem and progenitor cells (HSPCs) are largely unknown. Reviewers: H.D., Peking University; and L.S., University of North Carolina at Chapel Hill. During hematopoiesis, transcription factors (TFs) control in- The authors declare no conflict of interest. duction and maintenance of the expression of lineage-specific genes Published under the PNAS license. and suppression of competing gene expression of other lineages (8– 14). MkPs are originally derived from hematopoietic stem cells See Commentary on page 9818. 1 (HSCs) through a well-documented stepwise differentiation (15, 16). To whom correspondence may be addressed. Email: [email protected] or irv@stanford. edu. To date, only a few TFs have been reported to be involved in this 2Present addresses: Medical Sciences Innovation Hub Program, RIKEN, Nihonbashi, 103- process, including AML1, FLI1, GABPA, GATA1, RUNX1, 0027 Tokyo, Japan; and Center for Integrative Medical Sciences, RIKEN, Yokohama, 230- NFE2, SCL, GATA2, MYB, and LMO2 (17–19). The bipotent 0045 Kanagawa, Japan. megakaryocyte-erythroid progenitors (MEPs) can directly give rise 3Present address: Department of Medicine and Biosystemic Science, Kyushu University to MkPs and erythroid progenitors (EPs), which further develop Graduate School of Medical Sciences, 812-8582 Fukuoka City, Japan. into megakaryocytes and erythrocytes, respectively (20). MkPs and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. EPs share some TFs, including GATA1, FOG1, SCL, and GFI1b, 1073/pnas.1805434115/-/DCSupplemental. but also have several lineage-specific genes, such as KLF1 (erythroid) Published online August 27, 2018. E9308–E9316 | PNAS | vol. 115 | no. 40 www.pnas.org/cgi/doi/10.1073/pnas.1805434115 Downloaded by guest on September 25, 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 SEE COMMENTARY 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
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