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Stem Cells

In vitro expansion of CD34+CD38− cells under stimulation with hematopoietic growth factors on AGM-S3 cells in juvenile myelomonocytic leukemia

Leukemia volume 29, pages 606–614 (2015)Cite this article

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Abstract

Using serum-containing culture, we examined whether AGM-S3 stromal cells, alone or in combination with hematopoietic growth factor(s), stimulated the proliferation of CD34+ cells from patients with juvenile myelomonocytic leukemia (JMML). AGM-S3 cells in concert with stem cell factor plus thrombopoietin increased the numbers of peripheral blood CD34+ cells to approximately 20-fold of the input value after 2 weeks in nine JMML patients with either PTPN11 mutations or RAS mutations, who received allogeneic hematopoietic transplantation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) also augmented the proliferation of JMML CD34+ cells on AGM-S3 cells. The expansion potential of CD34+ cells was markedly low in four patients who achieved spontaneous hematological improvement. A large proportion of day-14-cultured CD34+ cells were negative for CD38 and cryopreservable. Cultured JMML CD34+CD38− cells expressed CD117, CD116, c-mpl, CD123, CD90, but not CXCR4, and formed GM and erythroid colonies. Day-7-cultured CD34+ cells from two of three JMML patients injected intrafemorally into immunodeficient mice stimulated with human GM-CSF after transplantation displayed significant hematopoietic reconstitution. The abilities of OP9 cells and MS-5 cells were one-third and one-tenth, respectively, of the value obtained with AGM-S3 cells. Our culture system may provide a useful tool for elucidating leukemogenesis and for therapeutic approaches in JMML.

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References

  1. Koike K, Matsuda K . Recent advances in the pathogenesis and management of juvenile myelomonocytic leukaemia. Br J Haematol 2008; 141: 567–575.

    Article  CAS  PubMed  Google Scholar 

  2. Loh ML . Recent advances in the pathogenesis and treatment of juvenile myelomonocytic leukaemia. Br J Haematol 2011; 152: 677–687.

    Article  CAS  PubMed  Google Scholar 

  3. Sakaguchi H, Okuno Y, Muramatsu H, Yoshida K, Shiraishi Y, Takahashi M et al. Exome sequencing identifies secondary mutations of SETBP1 and JAK3 in juvenile myelomonocytic leukemia. Nat Genet 2013; 45: 937–941.

    Article  CAS  PubMed  Google Scholar 

  4. Sawai N, Koike K, Higuchi T, Ogami K, Oda M . Thrombopoietin enhances the production of myeloid cells, but not megakaryocytes, in juvenile chronic myelogenous leukemia. Blood 1998; 91: 4065–4073.

    CAS  PubMed  Google Scholar 

  5. Lapidot T, Grunberger T, Vormoor J, Estrov Z, Kollet O, Bunin N et al. Identification of human juvenile chronic myelogenous leukemia stem cells capable of initiating the disease in primary and secondary SCID mice. Blood 1996; 88: 2655–2664.

    CAS  PubMed  Google Scholar 

  6. Xu MJ, Tsuji K, Ueda T, Mukouyama YS, Hara T, Yang FC et al. Stimulation of mouse and human primitive hematopoiesis by murine embryonic aorta-gonad-mesonephros-derived stromal cell lines. Blood 1998; 92: 2032–2040.

    CAS  PubMed  Google Scholar 

  7. Ma F, Kambe N, Wang D, Shinoda G, Fujino H, Umeda K et al. Direct development of functionally mature tryptase/chymase double-positive connective tissue-type mast cells from primate embryonic stem cells. Stem Cells 2008; 26: 706–714.

    Article  CAS  PubMed  Google Scholar 

  8. Matsuda K, Sakashita K, Taira C, Tanaka-Yanagisawa M, Yanagisawa R, Shiohara M et al. Quantitative assessment of PTPN11 or RAS mutations at the neonatal period and during the clinical course in patients with juvenile myelomonocytic leukaemia. Br J Haematol 2010; 148: 593–599.

    Article  CAS  PubMed  Google Scholar 

  9. Sakashita K, Koike K, Kinoshita T, Shiohara M, Kamijo T, Taniguchi S et al. Dynamic DNA methylation change in the CpG island region of p15 during human myeloid development. J Clin Invest 2001; 108: 1195–1204.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Sawai N, Koike K, Ito S, Mwamtemi HH, Kurokawa Y, Kinoshita T et al. Neutrophilic cell production by combination of stem cell factor and thrombopoietin from CD34(+) cord blood cells in long-term serum-deprived liquid culture. Blood 1999; 93: 509–518.

    CAS  PubMed  Google Scholar 

  11. Ito M, Hiramatsu H, Kobayashi K, Suzue K, Kawahata M, Hioki K et al. NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells. Blood 2002; 100: 3175–3182.

    Article  CAS  PubMed  Google Scholar 

  12. Hiramatsu H, Nishikomori R, Heike T, Ito M, Kobayashi K, Katamura K et al. Complete reconstitution of human lymphocytes from cord blood CD34+ cells using the NOD/SCID/gammacnull mice model. Blood 2003; 102: 873–880.

    Article  CAS  PubMed  Google Scholar 

  13. Mazurier F, Doedens M, Gan OI, Dick JE . Rapid myeloerythroid repopulation after intrafemoral transplantation of NOD-SCID mice reveals a new class of human stem cells. Nat Med 2003; 9: 959–963.

    Article  CAS  PubMed  Google Scholar 

  14. Matsuda K, Shimada A, Yoshida N, Ogawa A, Watanabe A, Yajima S et al. Spontaneous improvement of hematologic abnormalities in patients having juvenile myelomonocytic leukemia with specific RAS mutations. Blood 2007; 109: 5477–5480.

    Article  CAS  PubMed  Google Scholar 

  15. Matsuda K, Matsuzaki S, Miki J, Hidaka E, Yanagisawa R, Nakazawa Y et al. Chromosomal change during 6-mercaptopurine (6-MP) therapy in juvenile myelomonocytic leukemia: the growth of a 6-MP-refractory clone that already exists at onset. Leukemia 2006; 20: 485–490.

    Article  CAS  PubMed  Google Scholar 

  16. Matsuda K, Yoshida N, Miura S, Nakazawa Y, Sakashita K, Hyakuna N et al. Long-term haematological improvement after non-intensive or no chemotherapy in juvenile myelomonocytic leukaemia and poor correlation with adult myelodysplasia spliceosome-related mutations. Br J Haematol 2012; 157: 647–650.

    Article  PubMed  Google Scholar 

  17. Yoshida N, Yagasaki H, Xu Y, Matsuda K, Yoshimi A, Takahashi Y et al. Correlation of clinical features with the mutational status of GM-CSF signaling pathway-related genes in juvenile myelomonocytic leukemia. Pediatr Res 2009; 65: 334–340.

    Article  CAS  PubMed  Google Scholar 

  18. Lapidot T, Kollet O . The essential roles of the chemokine SDF-1 and its receptor CXCR4 in human stem cell homing and repopulation of transplanted immune-deficient NOD/SCID and NOD/SCID/B2m(null) mice. Leukemia 2002; 16: 1992–2003.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by Grants-in-Aid for Scientific Research (No. 24390260 and No. 22591154) from the Ministry of Education of Japan and grant from the Preventive Medical Center of Shinshu University Hospital.

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Authors and Affiliations

  1. Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, Japan

    K Sakashita, S Saito, K Hirabayashi, T Kurata, L T N Uyen, Y Nakazawa & K Koike

  2. Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    I Kato, T Daifu, S Saida, H Hiramatsu & T Heike

  3. Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan

    Y Nishinaka & T Nakahata

  4. Department of Pediatric Hematology/Oncology, Research Hospital, Institute of Medical Science, University of Tokyo, Minato-ku, Japan,

    Y Ebihara & K Tsuji

  5. Division of Stem Cell Processing, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, Minato-ku, Japan,

    Y Ebihara, F Ma & K Tsuji

  6. Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China

    F Ma

  7. Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan

    K Matsuda

  8. Department of Pediatrics, Shinshu Ueda Medical Center, National Hospital Organization, Ueda, Japan,

    K Tsuji

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  1. K Sakashita
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Correspondence to K Koike.

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Sakashita, K., Kato, I., Daifu, T. et al. In vitro expansion of CD34+CD38− cells under stimulation with hematopoietic growth factors on AGM-S3 cells in juvenile myelomonocytic leukemia. Leukemia 29, 606–614 (2015). https://doi.org/10.1038/leu.2014.239

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