Open AccessMolecular Analysis of Neutrophil Differentiation from Human Induced Pluripotent Stem Cells Delineates the Kinetics of Key Regulators of Hematopoiesis
Author(s) -
Sweeney Colin L.,
Teng Ruifeng,
Wang Hongmei,
Merling Randall K.,
Lee Janet,
Choi Uimook,
Koontz Sherry,
Wright Daniel G.,
Malech Harry L.
Publication year - 2016
Publication title -
stem cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.159
H-Index - 229
eISSN - 1549-4918
pISSN - 1066-5099
DOI - 10.1002/stem.2332
Subject(s) - biology , haematopoiesis , microbiology and biotechnology , runx1 , progenitor cell , stem cell , cd34 , myeloid , endothelial stem cell , induced pluripotent stem cell , cellular differentiation , embryoid body , immunology , embryonic stem cell , genetics , in vitro , gene
A bstract 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/EBPα, 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/EBPɛ 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. S tem C ells 2016;34:1513–1526