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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

Hematopoietic stem cell expansion“SALL4”

Presented by: Shenbin

2012-10-21


Spalt sall
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



Hematopoietic stem cells
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.


Sall4
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


Role of sall4 in hematopoiesis
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


In vitro

In vivo

lentivirus

TAT-His-SALL4B Protein

Murine system

Nonhuman primate


Sall4 lentiviral infection of human cd34 cells
SALL4-Lentiviral infection of human CD34 hematopoietic stem cells ??+ 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


Transfused with GFP lentivirus hematopoietic stem cells ??

Transfused with SALL4B-GFP lentivirus

Aguila JR, Liao W, Yang J, et al. Blood 2011; 118:576–585.



Isolated CD34+ cell from peripheral blood of surviving and expanding

7 days after transfected with sall4b lentivirus

Aguila JR, Liao W, Yang J, et al. Blood 2011; 118:576–585.


75% less cytokines of surviving and expanding

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.


14 days of surviving and expanding

SALL4A vs. control = 368-fold

SALL4B vs. control = 384-fold

Aguila JR, Liao W, Yang J, et al. Blood 2011; 118:576–585.


Group SALL4A: 31 days later of surviving and expanding

Group SALL4B: 31 days later

Group control: 3 days later

Aguila JR, Liao W, Yang J, et al. Blood 2011; 118:576–585.


  • 14 days of culture of surviving and expanding

    • 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.



Tat sall4b protein induced cd34 cells expansion
TAT-SALL4B protein induced CD34+ cells expansion of surviving and expanding

  • 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.


Culture for 3-4 days of surviving and expanding

Aguila JR, Liao W, Yang J, et al. Blood 2011; 118:576–585.


Murine system
Murine system of surviving and expanding

  • 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)


Murine system1
Murine system of surviving and expanding

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


Transplantation
Transplantation of surviving and expanding

  • 10 days cell culture

  • Cell number: 2*10^6

  • Donor: Ly5.2+; recipient: Ly5.1 mice

  • Intravenously

  • Sublethally irradiated(500 rad)


The population of donor cells (Ly5.2+) in the of surviving and expandingperipheral 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


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


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


  • 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


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


Why? regenerated lineages contributed from donor cells

  • Epigenetic mechanisms involved in the regulatory functions of sall4 in hematopoietic stem/progenitor cells


  • SALL4-mediated activation of regenerated lineages contributed from donor cellsBmi-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.


Up to now regenerated lineages contributed from donor cells

  • 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


Prospect
Prospect regenerated lineages contributed from donor cells

  • 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


Thanks for your attention
Thanks for your attention regenerated lineages contributed from donor cells


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