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
Ronald P.Kuhnlein, Gotz Frommer, et al. The EMBO Journal vol. 13 no. 1
pp. 168 - 179, 1994
• 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
J Kohlhase, L Schubert, M Liebers et al. J Med Genet 2003;40:473–478
Research development of this field
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
• 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
 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
maintain the 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
• SALL4 is a robust stimulator for the
expansion of hematopoietic stem
cells ??
In vitro
lentivirus
TAT-His-SALL4B Protein
In vivo
Murine system
Nonhuman
primate
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
Transfused with GFP lentivirus
Transfused with SALL4B-GFP lentivirus
Aguila JR, Liao W, Yang J, et al. Blood 2011; 118:576–585.
• 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
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.
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.
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.
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.
• 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.
[4] Yang J, Gao C, Chai L, Ma Y. PLoS One 2010; 5:e10766
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.
Culture for 3-4 days
Aguila JR, Liao W, Yang J, et al. Blood 2011; 118:576–585.
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)
Murine system
Yang J, Aguila JR, Alipio Z, et al. J Hematol Oncol 2011; 4:38
Transplantation
•
•
•
•
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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 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
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?
• Epigenetic mechanisms involved in the
regulatory functions of sall4 in hematopoietic
stem/progenitor cells
• 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.
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
Prospect
• Nonhuman primate system
• Human system
– Clinical test
 Expanded HSC application
 Red blood cells
 Platelet
 Granulocyte
 Liver cells
 Beta cell
 …
attempt
TAT-His-SALL4B protein
Enucleation
Mechanisms
Confirmation; mechanisms; applications; Technical difficulties
Thanks for your attention