Xuechong Hong

Smooth muscle cells differentiated from reprogrammed embryonic lung fibroblasts through DKK3 signaling are potent for tissue engineering of vascular grafts.

Rationale: Smooth muscle cells (SMCs) are a key component of tissue-engineered vessels. However, the sources by which they can be isolated are limited. Objective: We hypothesized that a large number of SMCs could be obtained by direct reprogramming of fibroblasts, that is, direct differentiation of specific cell lineages before the cells reaching the pluripotent state. Methods and Results: We designed a combined protocol of reprogramming and differentiation of human neonatal lung fibroblasts. Four reprogramming factors (OCT4, SOX2, KLF4, and cMYC) were overexpressed in fibroblasts under reprogramming conditions for 4 days with cells defined as partially-induced pluripotent stem (PiPS) cells. PiPS cells did not form tumors in vivo after subcutaneous transplantation in severe combined immunodeficiency mice and differentiated into SMCs when seeded on collagen IV and maintained in differentiation media. PiPS-SMCs expressed a panel of SMC markers at mRNA and protein levels. Furthermore, the gene dickkopf 3 was found to be involved in the mechanism of PiPS-SMC differentiation. It was revealed that dickkopf 3 transcriptionally regulated SM22 by potentiation of Wnt signaling and interaction with Kremen1. Finally, PiPS-SMCs repopulated decellularized vessel grafts and ultimately gave rise to functional tissue-engineered vessels when combined with previously established PiPS-endothelial cells, leading to increased survival of severe combined immunodeficiency mice after transplantation of the vessel as a vascular graft. Conclusions: We developed a protocol to generate SMCs from PiPS cells through a dickkopf 3 signaling pathway, useful for generating tissue-engineered vessels. These findings provide a new insight into the mechanisms of SMC differentiation with vast therapeutic potential.

c-Kit+ progenitors generate vascular cells for tissue-engineered grafts through modulation of the Wnt/Klf4 pathway

The development of decellularised scaffolds for small diameter vascular grafts is hampered by their limited patency, due to the lack of luminal cell coverage by endothelial cells (EC) and to the low tone of the vessel due to absence of a contractile smooth muscle cells (SMC). In this study, we identify a population of vascular progenitor c-Kit+/Sca-1- cells available in large numbers and derived from immuno-privileged embryonic stem cells (ESCs). We also define an efficient and controlled differentiation protocol yielding fully to differentiated ECs and SMCs in sufficient numbers to allow the repopulation of a tissue engineered vascular graft. When seeded ex vivo on a decellularised vessel, c-Kit+/Sca-1-derived cells recapitulated the native vessel structure and upon in vivo implantation in the mouse, markedly reduced neointima formation and mortality, restoring functional vascularisation. We showed that Krüppel-like transcription factor 4 (Klf4) regulates the choice of differentiation pathway of these cells through β-catenin activation and was itself regulated by the canonical Wnt pathway activator lithium chloride. Our data show that ESC-derived c-Kit+/Sca-1-cells can be differentiated through a Klf4/β-catenin dependent pathway and are a suitable source of vascular progenitors for the creation of superior tissue-engineered vessels from decellularised scaffolds.

Mesenchymal stem cells and vascular regeneration

In recent years, MSCs have emerged as a promising therapeutic cell type in regenerative medicine. They hold great promise for treating cardiovascular diseases, such as myocardial infarction and limb ischemia. MSCs may be utilized in both cell‐based therapy and vascular graft engineering to restore vascular function, thereby providing therapeutic benefits to patients. The efficacy of MSCs lies in their multipotent differentiation ability toward vascular smooth muscle cells, endothelial cells and other cell types, as well as their capacity to secrete various trophic factors, which are potent in promoting angiogenesis, inhibiting apoptosis and modulating immunoreaction. Increasing our understanding of the mechanisms of MSC involvement in vascular regeneration will be beneficial in boosting present therapeutic approaches and developing novel ones to treat cardiovascular diseases. In this review, we aim to summarize current progress in characterizing the in vivo identity of MSCs, to discuss mechanisms involved in cell‐based therapy utilizing MSCs, and to explore current and future strategies for vascular regeneration.

Resveratrol-Induced Vascular Progenitor Differentiation towards Endothelial Lineage via MiR-21/Akt/β-Catenin Is Protective in Vessel Graft Models

Background and Purpose Vessel graft failure is typically associated with arteriosclerosis, in which endothelial dysfunction/damage is a key event. Resveratrol has been shown to possess cardioprotective capacity and to reduce atherosclerosis. We aimed to study the influence of resveratrol on the behavior of resident stem cells that may contribute to graft arteriosclerosis. Experimental Approach Vascular resident progenitor cells and embryonic stem cells were treated with resveratrol under differentiating conditions and endothelial markers expression was evaluated. Expression of miR-21 and β-catenin was also tested and exogenously modified. Effects of resveratrol treatment in an ex vivo re-endothelialization model and on mice undergone vascular graft were evaluated. Key Results Resveratrol induced expression of endothelial markers such as CD31, VE-cadherin and eNOS in both progenitor and stem cells. We demonstrated that resveratrol significantly reduced miR-21 expression, which in turn reduced Akt phosphorylation. This signal cascade diminished the amount of nuclear β-catenin, inducing endothelial marker expression and increasing tube-like formation by progenitor cells. Both the inhibition of miR-21 and the knockdown of β-catenin were able to recapitulate the effect of resveratrol application. Ex vivo, progenitor cells treated with resveratrol produced better endothelialization of the decellularized vessel. Finally, in a mouse model of vessel graft, a resveratrol-enhanced diet was able to reduce lesion formation. Conclusions and Implications We provide the first evidence that oral administration of resveratrol can reduce neointimal formation in a model of vascular graft and elucidated the underpinning miR-21/Akt/β-catenin dependent mechanism. These findings may support the beneficial effect of resveratrol supplementation for graft failure prevention.

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Transdifferentiated Human Vascular Smooth Muscle Cells are a New Potential Cell Source for Endothelial Regeneration

Endothelial dysfunction is widely implicated in cardiovascular pathological changes and development of vascular disease. In view of the fact that the spontaneous endothelial cell (EC) regeneration is a slow and insufficient process, it is of great interest to explore alternative cell sources capable of generating functional ECs. Vascular smooth muscle cell (SMC) composes the majority of the vascular wall and retains phenotypic plasticity in response to various stimuli. The aim of this study is to test the feasibility of the conversion of SMC into functional EC through the use of reprogramming factors. Human SMCs are first dedifferentiated for 4 days to achieve a vascular progenitor state expressing CD34, by introducing transcription factors OCT4, SOX2, KLF4 and c-MYC. These SMC-derived progenitors are then differentiated along the endothelial lineage. The SMC-converted ECs exhibit typical endothelial markers expression and endothelial functions in vitro, in vivo and in disease model. Further comprehensive analysis indicates that mesenchymal-to-epithelial transition is requisite to initiate SMCs reprogramming into vascular progenitors and that members of the Notch signalling pathway regulate further differentiation of the progenitors into endothelial lineage. Together, we provide the first evidence of the feasibility of the conversion of human SMCs towards endothelial lineage through an intermediate vascular progenitor state induced by reprogramming.

Reprogramming towards endothelial cells for vascular regeneration

Endothelial damage and dysfunction are implicated in cardiovascular pathological changes and the development of vascular diseases. In view of the fact that the spontaneous endothelial cell (EC) regeneration is a slow and insufficient process, it is of great significance to explore alternative cell sources capable of generating functional ECs to repair damaged endothelium. Indeed, recent achievements of cell reprogramming to convert somatic cells to other cell types provide new powerful approaches to study endothelial regeneration. Based on progress in the research field, the present review aims to summarize the strategies and mechanisms of generating endothelial cells through reprogramming from somatic cells, and to examine what this means for the potential application of cell therapy in the clinic.

Smooth muscle cells differentiated from mesenchymal stem cells are regulated by microRNAs and suitable for vascular tissue grafts

Tissue-engineered vascular grafts with long-term patency are greatly needed in the clinical settings, and smooth muscle cells (SMCs) are a critical graft component. Human mesenchymal stem cells (MSCs) are used for generating SMCs, and understanding the underlying regulatory mechanisms of the MSC-to-SMC differentiation process could improve SMC generation in the clinic. Here, we found that in response to stimulation of transforming growth factor-β1 (TGFβ1), human umbilical cord–derived MSCs abundantly express the SMC markers α-smooth muscle actin (αSMA), smooth muscle protein 22 (SM22), calponin, and smooth muscle myosin heavy chain (SMMHC) at both gene and protein levels. Functionally, MSC-derived SMCs displayed contracting capacity in vitro and supported vascular structure formation in the Matrigel plug assay in vivo. More importantly, SMCs differentiated from human MSCs could migrate into decellularized mouse aorta and give rise to the smooth muscle layer of vascular grafts, indicating the potential of utilizing human MSC-derived SMCs to generate vascular grafts. Of note, microRNA (miR) array analysis and TaqMan microRNA assays identified miR-503 and miR-222-5p as potential regulators of MSC differentiation into SMCs at early time points. Mechanistically, miR-503 promoted SMC differentiation by directly targeting SMAD7, a suppressor of SMAD-related, TGFβ1-mediated signaling pathways. Moreover, miR-503 expression was SMAD4-dependent. SMAD4 was enriched at the miR-503 promoter. Furthermore, miR-222-5p inhibited SMC differentiation by targeting and down-regulating ROCK2 and αSMA. In conclusion, MSC differentiation into SMCs is regulated by miR-503 and miR-222-5p and yields functional SMCs for use in vascular grafts.

Adventitial Sca1+ Cells Transduced With ETV2 Are Committed to the Endothelial Fate and Improve Vascular Remodeling After Injury

Objective— Vascular adventitial Sca1+ (stem cell antigen-1) progenitor cells preferentially differentiate into smooth muscle cells, which contribute to vascular remodeling and neointima formation in vessel grafts. Therefore, directing the differentiation of Sca1+ cells toward the endothelial lineage could represent a new therapeutic strategy against vascular disease. Approach and Results— We thus developed a fast, reproducible protocol based on the single-gene transfer of ETV2 (ETS variant 2) to differentiate Sca1+ cells toward the endothelial fate and studied the effect of cell conversion on vascular hyperplasia in a model of endothelial injury. After ETV2 transduction, Sca1+ adventitial cells presented a significant increase in the expression of early endothelial cell genes, including VE-cadherin, Flk-1, and Tie2 at the mRNA and protein levels. ETV2 overexpression also induced the downregulation of a panel of smooth muscle cell and mesenchymal genes through epigenetic regulations, by decreasing the expression of DNA-modifying enzymes ten-eleven translocation dioxygenases. Adventitial Sca1+ cells grafted on the adventitial side of wire-injured femoral arteries increased vascular wall hyperplasia compared with control arteries with no grafted cells. Arteries seeded with ETV2-transduced cells, on the contrary, showed reduced hyperplasia compared with control. Conclusions— These data give evidence that the genetic manipulation of vascular progenitors is a promising approach to improve vascular function after endothelial injury.

222 Generation of Functional Endothelial-Like Cells Enabling Therapeutic Angiogenesis from Human Smooth Muscle Cells

Introduction Endothelial damage/dysfunction is a key event in cardiovascular pathological changes and development of ischaemic vascular disease, which is often followed by smooth muscle cell (SMC) proliferation and accumulation. If we could arbitrarily convert SMCs into endothelial cells (ECs), they may represent a promising new approach to achieve therapeutic angiogensis of ischaemic area. To test this hypothesis, the aim of the present study is to transdifferentiate human SMCs into functional ECs. Methods and results Human SMCs were first reprogrammed for 4 days to obtain a partially converted state, i.e. vascular progenitor, by introducing four transcription factors: Oct4, Sox2, Klf4 and c-Myc. These vascular progenitors were then subjected to defined media and culture conditions to induce endothelial lineage differentiation. The differentiated cells expressed EC markers such as CD31, CD144, and eNOS at the mRNA and protein level. Next, CD34 positive cells were selected from the heterogeneous population of vascular progenitors and further cultured in endothelial inducing conditions. The CD34+ cells were able to give rise to a more homogenous endothelial-like population in higher efficiency. The converted ECs displayed typical endothelial functional properties including acetylated-LDL uptake and tube formation in vitro and in vivo. More importantly, these ECs-derived from human SMCs exhibited therapeutic angiogenesis capacity which improved blood flow recovery when applied in the murine ischaemic hindlimb model. To explore the mechanisms involved in SMCs to ECs transdifferentiation, whole mRNAs from different reprogrammed cells were analysed by RNA-Sequencing techniques. Comprehensive analysis indicated that mesenchymal-to-epithelial transition was requisite to initiate SMCs reprogramming to vascular progenitor. Furthermore, it was demonstrated that hairy and enhancer of split 5 (HES5), a member of a family of basic helix-loop-helix transcriptional factor of the Notch signalling pathway, regulated progenitors to endothelial lineage differentiation. Conclusions In this study, we provided the first evidence of converting human SMCs towards functional EC lineage involving the mechanisms of mesenchymal-to-epithelial transition and the Notch signalling pathway. Furthermore, the converted ECs displayed a therapeutic capacity of angiogenesis in a murine ischaemic model.