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2016 ; 34
(6
): 1513-26
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Molecular Analysis of Neutrophil Differentiation from Human Induced Pluripotent
Stem Cells Delineates the Kinetics of Key Regulators of Hematopoiesis
#MMPMID26866427
Sweeney CL
; Teng R
; Wang H
; Merling RK
; Lee J
; Choi U
; Koontz S
; Wright DG
; Malech HL
Stem Cells
2016[Jun]; 34
(6
): 1513-26
PMID26866427
show ga
In vitro generation of mature neutrophils from human induced pluripotent stem
cells (iPSCs) requires hematopoietic progenitor development followed by myeloid
differentiation. The purpose of our studies was to extensively characterize this
process, focusing on the critical window of development between hemogenic
endothelium, hematopoietic stem/progenitor cells (HSPCs), and myeloid commitment,
to identify associated regulators and markers that might enable the stem cell
field to improve the efficiency and efficacy of iPSC hematopoiesis. We utilized a
four-stage differentiation protocol involving: embryoid body (EB) formation
(stage-1); EB culture with hematopoietic cytokines (stage-2); HSPC expansion
(stage-3); and neutrophil maturation (stage-4). CD34(+) CD45(-) putative
hemogenic endothelial cells were observed in stage-3 cultures, and expressed
VEGFR-2/Flk-1/KDR and VE-cadherin endothelial markers, GATA-2, AML1/RUNX1, and
SCL/TAL1 transcription factors, and endothelial/HSPC-associated microRNAs miR-24,
miR-125a-3p, miR-126/126*, and miR-155. Upon further culture, CD34(+) CD45(-)
cells generated CD34(+) CD45(+) HSPCs that produced hematopoietic CFUs.
Mid-stage-3 CD34(+) CD45(+) HSPCs exhibited increased expression of GATA-2,
AML1/RUNX1, SCL/TAL1, C/EBP?, and PU.1 transcription factors, but exhibited
decreased expression of HSPC-associated microRNAs, and failed to engraft in
immune-deficient mice. Mid-stage-3 CD34(-) CD45(+) cells maintained PU.1
expression and exhibited increased expression of hematopoiesis-associated
miR-142-3p/5p and a trend towards increased miR-223 expression, indicating
myeloid commitment. By late Stage-4, increased CD15, CD16b, and C/EBP? expression
were observed, with 25%-65% of cells exhibiting morphology and functions of
mature neutrophils. These studies demonstrate that hematopoiesis and neutrophil
differentiation from human iPSCs recapitulates many features of embryonic
hematopoiesis and neutrophil production in marrow, but reveals unexpected
molecular signatures that may serve as a guide for enhancing iPSC hematopoiesis.
Stem Cells 2016;34:1513-1526.