Abstract
Introduction:
A major roadblock for generating human pluripotent stem cell (hPSCs) derivatives highly enriched in cardiomyogenic precursors (CPCs), has been the lack of CPC-specific cell surface markers.
Hypothesis:
Based on observations that adult CPCs are responsive to growth hormone-releasing hormone (GHRH) signaling, we hypothesized that the GHRH receptor (GHRHR) is a specific cell-surface marker for hPSC-derived CPCs.
Methods:
We performed temporal analysis of GHRHR expression in an
in-vitro
model of human cardiogenesis using induced hPSCs (hiPSCs) and
SOX10::GFP
embryonic hPSCs (hESCs)
;
and mouse (
in-vivo
) cardiogenesis in wild-type (WT),
MEF2c-AHF-Cre, Wnt1-Cre2
and
cKit-CreERT2/+
reporter mice.
Results:
Gene expression and confocal immunofluorescence analyses during chemically-defined, stage-specific, cardiac lineage differentiation indicated that GHRHR is not expressed in undifferentiated hiPSCs or during specification into primitive streak-like Brachyury
+
or Mesp1
+
precardiac cells; but is induced in cardiogenic mesoderm-like cells, at the stage of commitment into NKX2.5
+
and/or ISL1
+
CPCs (
p
=0.001) and persists in Troponin-T
+
cardiomyocytes. Similarly, experiments modeling cardiac neural crest (CNC) with
SOX10::GFP
hESCs indicated that GHRHR is not expressed by GFP
+
CNCs but is induced following differentiation into NKX2.5
+
and/or ISL1
+
derivatives. Importantly, stimulation with 1μm recombinant GHRH during days 5-7 of hiPSCs differentiation increased
NKX2.5
expression 2.5-fold, an effect that was abolished by exposure to 1μM Somatostatin, a GHRH antagonist (
p
=0.0009). Last, in vivo analyses in WT
, MEF2c-AHF-Cre, Wnt1-Cre2
and
cKit-CreERT2/+
reporter embryonic and postnatal hearts corroborated that GHRHR specifically marks NKX2.5
+
mesoderm- and CNC-lineage descendants in vivo, whereas GHRHR is not expressed by
Wnt1-Cre2
and
cKit-CreERT2/+
CNCs descendants that are Nkx2.5
–
.
Conclusions:
Together these findings indicate that GHRHR is universally expressed by NKX2.5
+
/ISL1
+
CPCs and cardiomyocytes of both mesoderm and CNC origin. Therefore, GHRHR appears to be a valuable cell-surface marker for the selection and enrichment of CPCs from hPSCs for biomedical and regenerative medicine applications.