Specific marking of hESCs-derived hematopoietic lineage by WAS-promoter driven lentiviral vectors.
Genetic manipulation of human embryonic stem cells (hESCs) is instrumental for tracing lineage commitment and to studying human development. Here we used hematopoietic-specific Wiskott-Aldrich syndrome gene (WAS)-promoter driven lentiviral vectors (LVs) to achieve highly specific gene expression in hESCs-derived hematopoietic cells. We first demonstrated that endogenous WAS gene was not expressed in undifferentiated hESCs but was evident in hemogenic progenitors (CD45(-)CD31(+)CD34(+)) and hematopoietic cells (CD45(+)). Accordingly, WAS-promoter driven LVs were unable to express the eGFP transgene in undifferentiated hESCs. eGFP(+) cells only appeared after embryoid body (EB) hematopoietic differentiation. The phenotypic analysis of the eGFP(+) cells showed marking of different subpopulations at different days of differentiation. At days 10-15, AWE LVs tag hemogenic and hematopoietic progenitors cells (CD45(-)CD31(+)CD34(dim) and CD45(+)CD31(+)CD34(dim)) emerging from hESCs and at day 22 its expression became restricted to mature hematopoietic cells (CD45(+)CD33(+)). Surprisingly, at day 10 of differentiation, the AWE vector also marked CD45(-)CD31(low/-)CD34(-) cells, a population that disappeared at later stages of differentiation. We showed that the eGFP(+)CD45(-)CD31(+) population generate 5 times more CD45(+) cells than the eGFP(-)CD45(-)CD31(+) indicating that the AWE vector was identifying a subpopulation inside the CD45(-)CD31(+) cells with higher hemogenic capacity. We also showed generation of CD45(+) cells from the eGFP(+)CD45(-)CD31(low/-)CD34(-) population but not from the eGFP(-)CD45(-)CD31(low/-)CD34(-) cells. This is, to our knowledge, the first report of a gene transfer vector which specifically labels hemogenic progenitors and hematopoietic cells emerging from hESCs. We propose the use of WAS-promoter driven LVs as a novel tool to studying human hematopoietic development.
Human DNA Variability Department, Pfizer-Universidad de Granada-Junta de Andalucía Centre for Genomics and Oncological Research, GENYO, Granada, Spain.
Pilar
P
Muñoz
Miguel G
MG
Toscano
Pedro J
PJ
Real
Karim
K
Benabdellah
Marién
M
Cobo
Clara
C
Bueno
Verónica
V
Ramos-Mejía
Pablo
P
Menendez
Per
P
Anderson
Francisco
F
Martín
D001483:Base Sequence; D019070:Cell Lineage; D002478:Cells, Cultured; D017931:DNA Primers; D053595:Embryonic Stem Cells / Q000166:cytology*; D005434:Flow Cytometry; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic
D017931:DNA Primers