Conversion of Human Fibroblasts to Functional Endothelial Cells by Defined Factors

Abstract

Objective Transdifferentiation of fibroblasts to endothelial cells (ECs) may provide a novel therapeutic avenue for diseases including ischemia and fibrosis. Here we demonstrate that human fibroblasts can be transdifferentiated into functional ECs by using only two factors, Oct4 and Klf4, under inductive signaling conditions. Approach and Results To determine if human fibroblasts could be converted into ECs by transient expression of pluripotency factors, human neonatal fibroblasts were transduced with lentiviruses encoding Oct4 and Klf4 in the presence of soluble factors that promote the induction of an endothelial program. After 28 days, clusters of induced endothelial (iEnd) cells appeared and were isolated for further propagation and subsequent characterization. The iEnd cells resembled primary human ECs in their transcriptional signature by expressing endothelial phenotypic markers such as CD31, VE-cadherin, and von Willebrand Factor. Furthermore, the iEnd cells could incorporate acetylated low density lipoprotein, and form vascular structures in vitro and in vivo. When injected into the ischemic limb of mice, the iEnd cells engrafted, increased capillary density, and enhanced tissue perfusion. During the transdifferentiation process, the endogenous pluripotency network was not activated, suggesting that this process bypassed a pluripotent intermediate step. Conclusions Pluripotent factor–induced transdifferentiation can be successfully applied for generating functional autologous ECs for therapeutic applications.

Direct Conversion of Adult Skin Fibroblasts to Endothelial Cells by Defined Factors

“…8 The direct conversion of adult cells into ECs can provide new therapeutic modalities to overcome the hurdles associated factors (OCT4, SOX2, KLF4, and c-MYC) 24,25 can convert human fibroblasts into ECs without inducing pluripotent status. [30][31][32] However, because conversion by these techniques involves the generation of intermediate progenitor cells or partial iPSCs, these are essentially different from our direct conversion technology. Moreover, because these studies used pluripotent factors, there are still concerns about their tumorigenic potential.…”

Small Molecule Derived From Carboxyethylpyrrole Protein Adducts Promotes Angiogenesis in a Mouse Model of Peripheral Arterial Disease

“…After 14 days, the subcutaneous plugs were explanted, and immunostaining was performed using antibody against murine CD31 for identifying the microvessels. Some groups were treated with bFGF as a reference control because bFGF is known to induce an angiogenic response in subcutaneous Matrigel plugs 18. As shown in Figure 10, microvessel density was significantly increased by the treatment of CEP03 (27.6±14/mm 2 ) or bFGF (27.8±18.5/mm 2 ) compared with vehicle control mice that received only Matrigel (4.7±2.5/mm 2 ; P <0.05).…”

“…The animals were randomized to receive Matrigel alone (control), bFGF (6 nmol/L), CEP03 (2 mmol/L), or bFGF (6 nmol/L) plus CEP03 (2 mmol/L; n=8 per randomized group). Some groups were treated with bFGF as a reference control because bFGF is known to induce an angiogenic response in subcutaneous Matrigel plugs 18. All reagents were dissolved in Matrigel (Geltrex; Gibco) at 4°C and injected at 300 μL per injection subcutaneously to form a gelatinous plug.…”

Direct reprogramming of somatic cells: an update

“…Some pluripotency factors used to produce iPSC can directly reprogram some cell types such as cardiomyocytes (Efe et al, 2011), neural stem cells or progenitors , angioblast-like progenitor cells (Kurian et al, 2013), endothelial cells (Li et al, 2013a), pancreatic lineages , and hepatocytes . Ma et al (2013) showed that pluripotent factors can reprogram adult cells into pluripotent cells with multiple steps and that at certain steps some cells' fates are formed as transition stages of epigenetic reprogramming .…”