Efficient Direct Reprogramming of Mature Amniotic Cells into Endothelial Cells by ETS Factors and TGFβ Suppression

Abstract

ETS transcription factors ETV2, FLI1 and ERG1 specify pluripotent stem cells into endothelial cells (ECs). However, these ECs are unstable and drift towards non-vascular cell fates. We show that human mid-gestation c-Kit− lineage-committed amniotic cells (ACs) can be readily reprogrammed into induced vascular endothelial cells (iVECs). Transient ETV2 expression in ACs generated proliferative but immature iVECs, while co-expression with FLI1/ERG1 endowed iVECs with a vascular repertoire and morphology matching mature stable ECs. Brief TGFβ-inhibition functionalized VEGFR2 signaling, augmenting specification of ACs to iVECs. Genome-wide transcriptional analyses showed that iVECs are similar to adult ECs in which vascular-specific genes are turned on and non-vascular genes are silenced. Functionally, iVECs form long-lasting patent vasculature in Matrigel plugs and regenerating livers. Thus, short-term ETV2 expression and TGFβ-inhibition along with constitutive ERG1/FLI1 co-expression reprogram mature ACs into durable and functional iVECs with clinical-scale expansion potential. Public banking of HLA-typed iVECs would establish a vascular inventory for treatment of genetically diverse disorders.

Mouse Embryonic Stem Cells Have Underdeveloped Antiviral Mechanisms That Can Be Exploited for the Development of mRNA-Mediated Gene Expression Strategy

“…It is increasingly clear that obtaining ESC-derived cells with clinical quality by existing methods is a difficult task. Based on the same principle and methodology for iPSC reprogramming, a recent study demonstrated that forced expression of three transfection factors (Etv2, Fli1, and Erg1) that control vascular differentiation effectively converted hESCs to endothelial cells with significantly increased yield and maturity [23,45]. This study demonstrates that intervention at the transcriptional level can provide a strong internal driving force for cell-specific differentiation.…”

“…This study demonstrates that intervention at the transcriptional level can provide a strong internal driving force for cell-specific differentiation. However, the expression of the aforementioned transcription factors in that study was also mediated by viral vectors [23]. Using synthetic mRNA as an alternative gene expression approach could avoid the safety concerns associated with viral vectors.…”

“…It is conceivable that the inefficient differentiation of existing methods is, at least in part, due to insufficient transcription activation during in vitro differentiation. A recent study reported that hESCs can be effectively differentiated into endothelial cells by viral vectormediated expression of transcription factors that control vascular differentiation [23], demonstrating that intervention at the transcription level can provide a much stronger driving force for ESC differentiation into a specific cell fate.…”

“…To further determine the functionality and compatibility of synthetic mRNA with mESCs, we transfected D3 cells with the Etv2 mRNA (m 7 GpppA-Etv2-polyA), a member of the E-twenty-six (ETS) transcription factor family that directly transactivates several genes required for vascular development [23,45]. As shown in Fig.…”

Direct reprogramming of somatic cells: an update

“…Since 2000, several transcription factors were discovered and were successfully used to reprogram target cells such as pancreatic islet cells (Zhou et al, 2008), neurons (Fishman et al, 2015;Vierbuchen et al, 2010), hepatocytes (Huang et al, 2011;Sekiya and Suzuki, 2011), endothelial cells (Ginsberg et al, 2012;Han et al, 2014), smooth muscle cells (Karamariti et al, 2013), and hepatocyte like cells (Simeonov and Uppal, 2014).…”