Inhibition of MicroRNA-221 and 222 Enhances Hematopoietic Differentiation from Human Pluripotent Stem Cells via c-KIT Upregulation

  • Lee, Ji Yoon (Department of Biomedical Sciences, Stem Cell Institute, CHA University) ;
  • Kim, MyungJoo (Department of Internal Medicine, School of Medicine, Kangwon National University) ;
  • Heo, Hye-Ryeon (Department of Internal Medicine, School of Medicine, Kangwon National University) ;
  • Ha, Kwon-Soo (Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University) ;
  • Han, Eun-Taek (Department of Medical Environmental Biology and Tropical Medicine, School of Medicine, Kangwon National University) ;
  • Park, Won Sun (Department of Physiology, School of Medicine, Kangwon National University) ;
  • Yang, Se-Ran (Department of Thoracic & Cardiovascular Surgery, School of Medicine, Kangwon National University) ;
  • Hong, Seok-Ho (Department of Internal Medicine, School of Medicine, Kangwon National University)
  • Received : 2018.06.02
  • Accepted : 2018.10.10
  • Published : 2018.11.30


The stem cell factor (SCF)/c-KIT axis plays an important role in the hematopoietic differentiation of human pluripotent stem cells (hPSCs), but its regulatory mechanisms involving microRNAs (miRs) are not fully elucidated. Here, we demonstrated that supplementation with SCF increases the hematopoietic differentiation of hPSCs via the interaction with its receptor tyrosine kinase c-KIT, which is modulated by miR-221 and miR-222. c-KIT is comparably expressed in undifferentiated human embryonic and induced pluripotent stem cells. The inhibition of SCF signaling via treatment with a c-KIT antagonist (imatinib) during hPSC-derived hematopoiesis resulted in reductions in the yield and multi-lineage potential of hematopoietic progenitors. We found that the transcript levels of miR-221 and miR-222 targeting c-KIT were significantly lower in the pluripotent state than they were in terminally differentiated somatic cells. Furthermore, suppression of miR-221 and miR-222 in undifferentiated hPSC cultures induced more hematopoiesis by increasing c-KIT expression. Collectively, our data implied that the modulation of c-KIT by miRs may provide further potential strategies to expedite the generation of functional blood cells for therapeutic approaches and the study of the cellular machinery related to hematologic malignant diseases such as leukemia.


Supported by : National Research Foundation of Korea (NRF)


  1. Ambros, V. (2004). The functions of animal microRNAs. Nature 431, 350-355.
  2. Bartel, D.P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281-297.
  3. Bashamboo, A., Taylor, A.H., Samuel, K., Panthier, J.J., Whetton, A.D., and Forrester, L.M. (2006). The survival of differentiating embryonic stem cells is dependent on the SCF-KIT pathway. J. Cell Sci. 119, 3039-3046.
  4. Bhatia, M., Bonnet, D., Wu, D., Murdoch, B., Wrana, J., Gallacher, L., and Dick, J.E. (1999). Bone morphogenetic proteins regulate the developmental program of human hematopoietic stem cells. J. Exp. Med. 189, 1139-1148.
  5. Carow, C.E., Hangoc, G., Cooper, S.H., Williams, D.E., and Broxmeyer, H.E. (1991). Mast cell growth factor (c-kit ligand) supports the growth of human multipotential progenitor cells with a high replating potential. Blood 78, 2216-2221.
  6. Chadwick, K., Wang, L., Li, L., Menendez, P., Murdoch, B., Rouleau, A., and Bhatia, M. (2003). Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells. Blood 102, 906-915.
  7. Ding, L., Saunders, T.L., Enikolopov, G., and Morrison, S.J. (2012). Endothelial and perivascular cells maintain haematopoietic stem cells. Nature 481, 457-462.
  8. Felli, N., Fontana, L., Pelosi, E., Botta, R., Bonci, D., Facchiano, F., Liuzzi, F., Lulli, V., Morsilli, O., Santoro, S., et al. (2005). MicroRNAs 221 and 222 inhibit normal erythropoiesis and erythroleukemic cell growth via kit receptor down-modulation. P. Nat. Acad. Sci. USA 102, 18081-18086.
  9. Gao, X., Lin, J., Gao, L., Deng, A., Lu, X., Li, Y., Wang, L., and Yu, L. (2015). High expression of c-kit mRNA predicts unfavorable outcome in adult patients with t(8;21) acute myeloid leukemia. PloS one 10, e0124241.
  10. Gits, C.M., van Kuijk, P.F., Jonkers, M.B., Boersma, A.W., van Ijcken, W.F., Wozniak, A., Sciot, R., Rutkowski, P., Schöffski, P., Taguchi, T., et al.(2013). MiR-17-92 and miR-221/222 cluster members target KIT and ETV1 in human gastrointestinal stromal tumours. British J. Cancer 109,1625-35.
  11. Guo, Y., Graham-Evans, B., and Broxmeyer, H.E. (2006). Murine embryonic stem cells secrete cytokines/growth modulators that enhance cell survival/anti-apoptosis and stimulate colony formation of murine hematopoietic progenitor cells. Stem Cells 24, 850-856.
  12. Han, C.P., Lin, W.L., Wang, P.H., Yang, S.F., Lewis, J.S. Jr., Chen, C.K., Ruan, A., Kuo, J.F., Lee, M.Y., and Chiang, H. (2011). Overexpression of c-KIT (CD117) occurs infrequently in squamous cell carcinoma of the uterine cervix. Histopathology 58, 988-990.
  13. Hong, S.H., Rampalli, S., Lee, J.B., McNicol, J., Collins, T., Draper, J.S., and Bhatia, M. (2011). Cell fate potential of human pluripotent stem cells is encoded by histone modifications. Cell Stem Cell 9, 24-36.
  14. Kim, H., Cho, H.J., Kim, S.W., Liu, B., Choi, Y.J., Lee, J., Sohn, Y.D., Lee, M.Y., Houge, M.A., and Yoon, Y.S. (2010). $CD31^+$ cells represent highly angiogenic and vasculogenic cells in bone marrow: novel role of nonendothelial $CD31^+$ cells in neovascularization and their therapeutic effects on ischemic vascular disease. Circulation Res. 107, 602-614.
  15. Kim, W.J., Lim, J.H., Hong, Y., Hong, S.H., Bang, C.Y., Lee, J.S., Oh, Y.M., and Kim, J.H. (2017). Altered miRNA expression in lung tissues of patients with chronic obstructive pulmonary disease. Mol. Cell Toxicol. 13, 207-212.
  16. Kimura, Y., Ding, B., Imai, N., Nolan, D.J., Butler, J.M., and Rafii, S. (2011). c-Kit-mediated functional positioning of stem cells to their niches is essential for maintenance and regeneration of adult hematopoiesis. PloS one 6, e26918.
  17. Lancrin, C., Sroczynska, P., Stephenson, C., Allen, T., Kouskoff, V., and Lacaud, G. (2009). The haemangioblast generates haematopoietic cells through a haemogenic endothelium stage. Nature 457, 892-895.
  18. Lim, L.P., Lau, N.C., Garrett-Engele, P., Grimson, A., Schelter, J.M., Castle, J., Bartel, D.P., Linsley, P.S., and Johnson, J.M. (2005). Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433, 769-773
  19. Lee, J.E., Kang, M.S., Park, M.H., Shim, S.H., Yoon, T.K., Chung, H.M., and Lee, D.R. (2010). Evaluation of 28 human embryonic stem cell lines for use as unrelated donors in stem cell therapy: implications of HLA and ABO genotypes. Cell Transplantation 19, 1383-1395.
  20. Lis, R., Karrasch, C.C., Poulos, M.G., Kunar, B., Redmond, D., Duran, J.G.B., Badwe, C.R., Schachterle, W., Ginsberg, M., Xiang, J., et al. (2017). Conversion of adult endothelium to immunocompetent haematopoietic stem cells. Nature 545, 439-445.
  21. Lu, M., Glover, C.H., Tien, A.H., Humphries, R.K., Piret, J.M., and Helgason, C.D. (2007). Involvement of tyrosine kinase signaling in maintaining murine embryonic stem cell functionality. Exp. Hematol. 35, 1293-1302.
  22. Marshall, C.J., Sinclair, J.C., Thrasher, A.J., and Kinnon, C. (2007). Bone morphogenetic protein 4 modulates c-Kit expression and differentiation potential in murine embryonic aorta-gonad-mesonephros haematopoiesis in vitro. British J. Haematol. 139, 321-330.
  23. Moses, B.S., Evans, R., Slone, W.L., Piktel, D., Martinez, I., Craig, M.D., and Gibson, L.F. (2016). Bone Marrow Microenvironment Niche Regulates miR-221/222 in Acute Lymphoblastic Leukemia. Molecular Cancer Research : MCR 14, 909-919.
  24. Ohwada, M., Wada, T., Saga, Y., Tsunoda, S., Jobo, T., Kuramoto, H., Konno, R., and Suzuki, M. (2006). C-kit overexpression in neuroendocrine small cell carcinoma of the uterine cervix. European J. Gynaecological Oncol. 27, 53-55.
  25. Perry, J.M., Harandi, O.F., and Paulson, R.F. (2007). BMP4, SCF, and hypoxia cooperatively regulate the expansion of murine stress erythroid progenitors. Blood 109, 4494-4502.
  26. Pick, M., Azzola, L., Mossman, A., Stanley, E.G., and Elefanty, A.G. (2007). Differentiation of human embryonic stem cells in serum-free medium reveals distinct roles for bone morphogenetic protein 4, vascular endothelial growth factor, stem cell factor, and fibroblast growth factor 2 in hematopoiesis. Stem Cells 25, 2206-2214.
  27. Poliseno, L., Tuccoli, A., Mariani, L., Evangelista, M., Citti, L., Woods, K., Mercatanti, A., Hammond, S., and Rainaldi, G. (2006). MicroRNAs modulate the angiogenic properties of HUVECs. Blood 108, 3068-3071.
  28. Ramon, L.A., Braza-Boils, A., Gilabert, J., Chirivella, M., Espana, F., Estelles, A., and Gilabert-Estelles, J. (2012). microRNAs related to angiogenesis are dysregulated in endometrioid endometrial cancer. Human Reproduction 27, 3036-3045.
  29. Rojas-Sutterlin, S., Lecuyer, E., and Hoang, T. (2014). Kit and Scl regulation of hematopoietic stem cells. Curr. Opin. Hematol. 21, 256-264.
  30. Rommer, A., Steinleitner, K., Hackl, H., Schneckenleithner, C., Engelmann, M., Scheideler, M., Vlatkovic, I., Kralovics, R., Cerny-Reiterer, S., Valent, P., et al. (2013). Overexpression of primary microRNA 221/222 in acute myeloid leukemia. BMC Cancer 13, 364.
  31. Spinello, I., Quaranta, M.T., Pasquini, L., Pelosi, E., Petrucci, E., Pagliuca, A., Castelli, G., Mariani, G., Diverio, D., Foa, R., et al. (2009). PLZF-mediated control on c-kit expression in CD34(+) cells and early erythropoiesis. Oncogene 28, 2276-2288.
  32. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872.
  33. Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. (1998). Embryonic stem cell lines derived from human blastocysts. Science 282, 1145-1147.
  34. Werbowetski-Ogilvie, T.E., Bosse, M., Stewart, M., Schnerch, A., Ramos-Mejia, V., Rouleau, A., Wynder, T., Smith, M.J., Dingwall, S., Carter, T., et al. (2009). Characterization of human embryonic stem cells with features of neoplastic progression. Nat. Biotechnol. 27, 91-97.
  35. Yang, Q.E., Racicot, K.E., Kaucher, A.V., Oatley, M.J., and Oatley, J.M. (2013). MicroRNAs 221 and 222 regulate the undifferentiated state in mammalian male germ cells. Development 140, 280-290.
  36. Zhang, P., Li, J., Tan, Z., Wang, C., Liu, T., Chen, L., Yong, J., Jiang, W., Sun, X., Du, L., et al. (2008). Short-term BMP-4 treatment initiates mesoderm induction in human embryonic stem cells. Blood 111, 1933-1941.