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Hematopoietic stem cell expansion “SALL4”

Hematopoietic stem cell expansion “SALL4”. Presented by: Shenbin 2012-10-21. Spalt SALL. Spalt(sal) homeotic gene function in the head and tail region of the Drosophila embryo mutations at SALL1 on chromosome 16q12. being associated with Townes-Brocks syndrome 耳前有附耳，皮赘，肛门闭锁、前移或形成直肠 - 肛门瘘

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Hematopoietic stem cell expansion “SALL4”

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  1. Hematopoietic stem cell expansion“SALL4” Presented by: Shenbin 2012-10-21

  2. SpaltSALL • Spalt(sal) homeotic gene function in the head and tail region of the Drosophila embryo • mutations at SALL1 on chromosome 16q12. being associated with Townes-Brocks syndrome • 耳前有附耳，皮赘，肛门闭锁、前移或形成直肠-肛门瘘 • mutations at SALL4 being shown to be causative in patients with Okihiro syndrome • 眼球后退综合征，一种先天性眼运动失调症 • 前臂畸形和耳聋,或者桡侧手畸形、眼肌运动受限 Ronald P.Kuhnlein, Gotz Frommer, et al. The EMBO Journal vol. 13 no. 1 pp. 168 - 179, 1994 J Kohlhase, L Schubert, M Liebers et al. J Med Genet 2003;40:473–478

  3. Research development of this field

  4. Hematopoietic stem cells • Self-renew • Capable of differentiating into all blood cell types[1] • Exist[2] • fetal bone marrow • adult bone marrow • peripheral blood • Liver • umbilical cord blood • Clinical[2] • Leukemia • inborn anomalies of the blood and immune system • Aplastic anemia(再生障碍性贫血) • Hemoglobinopathies (血红蛋白病) [1] Yang J, Aguila JR, Alipio Z, et al. J Hematol Oncol 2011; 4:38 [2] Aguila JR, Liao W, Yang J, et al. Blood 2011; 118:576–585.

  5. SALL4 • zinc-finger transcriptional factor[2] • SALL4 is one of four human homologues of the Drosophila region-specific homeotic gene spalt (sal) [3] http://en.wikipedia.org/wiki/Zinc_finger [2] Aguila JR, Liao W, Yang J, et al. Blood 2011; 118:576–585. [3] Jianchang Yangb, Wenbin Liao, et al. Curr Opin Hematol 2012, 19:287–291

  6. Role of SALL4 in hematopoiesis • Unlike many embryonic stemness factors, SALL4 is also expressed in adult stem cells, particularly hematopoietic precursor cells. • Overexpression of SALL4 is able to promote the proliferation and maintainthe self-renewal of (HSCs/HPCs). • Epigenetic mechanisms are likely involved in SALL4’s function in regulating the stem cell fate and self-renewal of HSCs/HPCs. • Dual functions of sall4 and its Regulated pathways in Hematopoiesis[3] • Suppress important differentiation genes • Activates key pluripotency genes • Interplay: • HOX family of transcription factors • the polycomb group (PcG) genes • components of the Wnt/b-catenin pathway • the Notch signaling pathway • bone morphogenic protein (BMP) family [3] Jianchang Yangb, Wenbin Liao, et al. Curr Opin Hematol 2012, 19:287–291

  7. SALL4 is a robust stimulator for the expansion of hematopoietic stem cells ?? In vitro In vivo lentivirus TAT-His-SALL4B Protein Murine system Nonhuman primate

  8. SALL4-Lentiviral infection of human CD34+ cells • Human bone marrow CD34+ cells • AllCells • Peripheral blood • Medium • StemSpan SFEM • StemCell Technologies • Stem Span SFEM+10%FBS+1% pen/strep(Invitrogen) • Cytokines • Flt-3: 100ng/ml • TPO: 100ng/ml • SCF: 100ng/ml • 12-well plate, 10^5 cells/well • Transduction • MOI: 10-20 • SALL4 lentivirus & GFP-control lentivirus • Infecte overnight for 12-15h

  9. Transfused with GFP lentivirus Transfused with SALL4B-GFP lentivirus Aguila JR, Liao W, Yang J, et al. Blood 2011; 118:576–585.

  10. To see whether the sall4-transduced cells were still capable of surviving and expanding • Culture in media containing 50% less cytokines • Culture in media containing 75% less cytokines

  11. Isolated CD34+ cell from peripheral blood 7 days after transfected with sall4b lentivirus Aguila JR, Liao W, Yang J, et al. Blood 2011; 118:576–585.

  12. 75% less cytokines 50000 cells 1st day Group SALL4A and SALL4B expanded approximately 130-fold Group control cells expanded 12-fold at most Aguila JR, Liao W, Yang J, et al. Blood 2011; 118:576–585.

  13. 14 days SALL4A vs. control = 368-fold SALL4B vs. control = 384-fold Aguila JR, Liao W, Yang J, et al. Blood 2011; 118:576–585.

  14. Group SALL4A: 31 days later Group SALL4B: 31 days later Group control: 3 days later Aguila JR, Liao W, Yang J, et al. Blood 2011; 118:576–585.

  15. 14 days of culture • Sall4-transduced cells growth and survival were independent of FLT-3L • Partially dependent of TPO • Dependent of SCF Marrow niche unlike high artificial levels in cell cultures Indicate SALL4 works in conjunction with the available cytokine levels in its environment to regulate cell expansion.

  16. [4] Yang J, Gao C, Chai L, Ma Y. PLoS One 2010; 5:e10766

  17. TAT-SALL4B protein induced CD34+ cells expansion • TAT-6His-SALL4B • E coli • Purified by nickel-nitrilotriacetic acid agarose • 200nM, twice a day Aguila JR, Liao W, Yang J, et al. Blood 2011; 118:576–585.

  18. Culture for 3-4 days Aguila JR, Liao W, Yang J, et al. Blood 2011; 118:576–585.

  19. Murine system • mouse HSC/HPC s (Lin-, Sca-1 +, c-Kit +; LSK cells) were isolated and transduced with lentiviruses carrying either GFP alone (control), or together with Sall4A or Sall4B isoform • (100 ng/ml mSCF, 6 ng/ml mIL-3 and 10 ng/ml hIL-6)

  20. Murine system Yang J, Aguila JR, Alipio Z, et al. J Hematol Oncol 2011; 4:38

  21. Transplantation • 10 days cell culture • Cell number: 2*10^6 • Donor: Ly5.2+; recipient: Ly5.1 mice • Intravenously • Sublethally irradiated(500 rad)

  22. The population of donor cells (Ly5.2+) in the peripheral blood of the recipient mice was examined by flow cytometry assays with the indicated antibodies at 2 weeks after BM transfusion. Peripheral blood mononuclear cells(PBMCs) Yang J, Aguila JR, Alipio Z, et al. J Hematol Oncol 2011; 4:38

  23. The Ly5.2+ cell percentages within the peripheral white blood cells of recipients were measured at 2, 20, and 50 weeks after transplantation of indicated lentivirus-infected BM cells. Error bars represent standard error of the mean. Yang J, Aguila JR, Alipio Z, et al. J Hematol Oncol 2011; 4:38

  24. The lineage (Gr-1, B220 and CD4/CD8) and primitive cell (Sca-1) distribution of Ly5.2+ cells was examined with the indicated antibodies for each group Yang J, Aguila JR, Alipio Z, et al. J Hematol Oncol 2011; 4:38

  25. Competitive repopulation assays to determine the activity of Sall4 transduced HSC/HPCs with day14 ex vivo cultured cells • Varying doses of cells baring Ly5.2 antigens were mixed with a constant number of non-transduced competitive BM cells (2*10^5, Ly5.1+), • injected into lethally irradiated (900 rad) congenic mice (Ly5.1). • 3 months

  26. Three months after transplantation, the percentage of regenerated lineages contributed from donor cells Yang J, Aguila JR, Alipio Z, et al. J Hematol Oncol 2011; 4:38

  27. Why? • Epigenetic mechanisms involved in the regulatory functions of sall4 in hematopoietic stem/progenitor cells

  28. SALL4-mediated activation of Bmi-1[5] • trimethylation of histone 3 lysine4 (H3K4), which is associated with gene activation • SALL4 can interact with an epigenetic repressor, Mi-2/nucleosome remodeling and deacetylase (NuRD) complex, mediating transcriptional repression[6] • PTEN, SALL4-immunocomplexes have histone deacetylase activity • SALL4 binds and actively recruits DNA methyltransferases (DNMTs), including DNMT1, DNMT3A, DNMT3B and DNMT3L, to target genes and represses their expression[7] [5] Yang J, Chai L, Liu F, et al. Bmi-1 is a target gene for SALL4 in hematopoietic and leukemic cells. Proc Natl Acad Sci U S A 2007; 104:10494–10499. [6] Lu J, Jeong H, Kong N, et al. Stem cell factor SALL4 represses the transcriptions of PTEN and SALL1 through an epigenetic repressor complex. PLoS One 2009; 4:e5577. [7] Yang J, Corsello TR, Ma Y. Stem cell gene SALL4 suppresses transcription through recruitment of DNA methyltransferases. J Biol Chem 2012; 287: 1996–2005.

  29. Up to now • Purchased normal human bone marrow CD34+ cells • Isolated human peripheral blood CD34+ cells • Isolated mouse bone marrow hematopoietic stem/progenitor cells(Lin-/Sca-1+/c-Kit+) • Isolated mouse peripheral blood HSCs • Myeloid progenitor cells(32D) • SALL4-GFP lentivirus transduct approach • TAT-His-SALL4B protein approach • Epigenetic mechanisms

  30. Prospect • Nonhuman primate system • Human system • Clinical test attempt TAT-His-SALL4B protein • Expanded HSC application • Red blood cells • Platelet • Granulocyte • Liver cells • Beta cell • … Enucleation Mechanisms Confirmation; mechanisms; applications; Technical difficulties

  31. Thanks for your attention

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