E Gara

Signaling Via PI3K/FOXO1A Pathway Modulates Formation and Survival of Human Embryonic Stem Cell-Derived Endothelial Cells

Vascular derivatives of human embryonic stem cells (hESC) are being developed as sources of tissue-specific cells for organ regeneration. However, identity of developmental pathways that modulate the specification of endothelial cells is not known yet. We studied phosphatidylinositol 3-kinase (PI3K)-Forkhead box O transcription factor 1A (FOXO1A) pathways during differentiation of hESC toward endothelial lineage and on proliferation, maturation, and cell death of hESC-derived endothelial cells (hESC-EC). During differentiation of hESC, expression of FOXO1A transcription factor was linked to the expression of a cluster of angiogenesis- and vascular remodeling-related genes. PI3K inhibitor LY294002 activated FOXO1A and induced formation of CD31(+) hESC-EC. In contrast, differentiating hESC with silenced FOXO1A by small interfering RNA (siRNA) showed lower mRNA levels of CD31 and angiopoietin2. LY294002 decreased proliferative activity of purified hESC-EC, while FOXO1A siRNA increased their proliferation. LY294002 inhibits migration and tube formation of hESC-EC; in contrast, FOXO1A siRNA increased in vitro tube formation activity of hESC-EC. After in vivo conditioning of cells in athymic nude rats, cells retain their low FOXO1A expression levels. PI3K/FOXO1A pathway is important for function and survival of hESC-EC and in the regulation of endothelial cell fate. Understanding these properties of hESC-EC may help in future applications for treatment of injured organs.

Morphology and vasoactive hormone profiles from endothelial cells derived from stem cells of different sources

Endothelial cells form a highly specialised lining of all blood vessels where they provide an anti-thrombotic surface on the luminal side and protect the underlying vascular smooth muscle on the abluminal side. Specialised functions of endothelial cells include their unique ability to release vasoactive hormones and to morphologically adapt to complex shear stress. Stem cell derived-endothelial cells have a growing number of applications and will be critical in any organ regeneration programme. Generally endothelial cells are identified in stem cell studies by well-recognised markers such as CD31. However, the ability of stem cell-derived endothelial cells to release vasoactive hormones and align with shear stress has not been studied extensively. With this in mind, we have compared directly the ability of endothelial cells derived from a range of stem cell sources, including embryonic stem cells (hESC-EC) and adult progenitors in blood (blood out growth endothelial cells, BOEC) with those cultured from mature vessels, to release the vasoconstrictor peptide endothelin (ET)-1, the cardioprotective hormone prostacyclin, and to respond morphologically to conditions of complex shear stress. All endothelial cell types, except hESC-EC, released high and comparable levels of ET-1 and prostacyclin. Under static culture conditions all endothelial cell types, except for hESC-EC, had the typical cobblestone morphology whilst hESC-EC had an elongated phenotype. When cells were grown under shear stress endothelial cells from vessels (human aorta) or BOEC elongated and aligned in the direction of shear. By contrast hESC-EC did not align in the direction of shear stress. These observations show key differences in endothelial cells derived from embryonic stem cells versus those from blood progenitor cells, and that BOEC are more similar than hESC-EC to endothelial cells from vessels. This may be advantageous in some settings particularly where an in vitro test bed is required. However, for other applications, because of low ET-1 release hESC-EC may prove to be protected from vascular inflammation.

Assessing the therapeutic readiness of stem cells for cardiovascular repair

Ischemic heart disease is among the most frequent causes of death and morbidity worldwide [1]. Despite advances in available cardiovascular therapies, a significant percentage of patients still die due to subsequent heart failure [2]. Regenerative medicine strategies to reduce myocardial injury and prevent left ventricular dysfunction are among the new advances in cardiovascular therapy. A quick internet search on ‘cardiac stem cell therapy’ may suggest that these approaches are currently part of routine treatment and are available for a wider patient population. However, our enthusiasm must be tempered given that whilst bone marrow transplantation is part of clinical regime in hematology, targeting solid organs with new cell therapy products is actually still in its early stage. Amongst others, guidelines such as the one by the International Society of Stem Cell Research in 2016 discussed current limitations of these applications and collected relevant recommendations [3]. The main hurdles to overcome before standard clinical application of stem cells are the immune rejection, risk for malignant transformation, and ethical concerns. On the technical side, detailed analyses of stem cells and standardization of cell sourcing, manufacturing, storage, delivery, and clinical intervention protocols are needed to validate stem cell therapy for routine clinical use (Figure 1).

FOXO1A modifies arterial and venous endothelial development from human pluripotent stem cells; they establish 3D vascular structures in vitro and quantifiable vascular networks in vivo

cells; they establish 3D vascular structures in vitro and quantifiable vascular networks in vivo Authors: E. Gara1, S.Z. Kiraly1, G. Kiszler2, J. Skopal1, M. Polos1, B. Merkely1, S.E. Harding3, G. Foldes3, 1Semmelweis University, Heart Center -Budapest Hungary, 2Semmelweis University, Department of Pathology and Experimental Cancer Research Budapest Hungary, 3Imperial College London, National Heart and Lung Institute London United Kingdom,Published on behalf of the European Society of Cardiology. All rights reserved.

Isolation, Expansion and Application of Human Mesenchymal Stem Cells

Human mesenchymal stem cells (hMSCs) are able to generate mesodermal derivatives, such as bone, cartilage, tendon, muscle, ligament and fat tissue; furthermore, they can differentiate to specific cell types, such as cardiomyocyte progenitors under defined conditions. Recently, their paracrine, pro-angiogenic, pro-myogenic, anti-apoptotic, anti-inflammatory and anti-fibrotic properties have been emphasized. These cells actively secrete a large number of bioactive molecules, peptides, hormones, and long non-coding RNAs. This may promote the regeneration of injured, ischemic tissues upon in vivo delivery. Human MSCs can be isolated from bone marrow, peripheral tissues such as adipose tissue, dental pulp, umbilical cord blood and matrix and potentially from peripheral blood. They have been widely used without the ethical issues implicated with pluripotent stem cell derivatives. Large randomized clinical trials underway with hMSCs show good safety and tolerability. In order to reach the number required for clinical use, efficient isolation and expansion of hMSCs are required. A variety of technologies are applied for hMSC expansion: static tissue culture flasks, cell factory and gas-permeable blood bags; or using dynamic culture by spinner flasks, stirred or rotary bioreactors with/without three-dimensional cell carriers. Routine quality controls are essential to maintain sterile culture conditions, ensure stable phenotype and monitor potential mutations before clinical applications. Human MSCs have recently been tested in large randomized clinical trials for cardiac regeneration. Although in vivo application was safe, clinical endpoints showed only neutral effects so far. In order to enhance regenerative capacity of hMSCs, cells may be boosted in vitro prior to implantation and delivery strategies and patient selection must also be improved.