Generation of heart and vascular system in rodents by blastocyst complementation.

Generating organs from stem cells through blastocyst complementation is a promising approach to meet the clinical need for transplants. In order to generate rejection-free organs, complementation of both parenchymal and vascular cells must be achieved, as endothelial cells play a key role in graft rejection. Here, we used a lineage-specific cell ablation system to produce mouse embryos unable to form both the cardiac and vascular systems. By mouse intraspecies blastocyst complementation, we rescued heart and vascular system development separately and in combination, obtaining complemented hearts with cardiomyocytes and endothelial cells of exogenous origin. Complemented chimeras were viable and reached adult stage, showing normal cardiac function and no signs of histopathological defects in the heart. Furthermore, we implemented the cell ablation system for rat-to-mouse blastocyst complementation, obtaining xenogeneic hearts whose cardiomyocytes were completely of rat origin. These results represent an advance in the experimentation towards the in vivo generation of transplantable organs.

Generation of an induced pluripotent stem cell line (ESi107-A) from a transthyretin amyloid cardiomyopathy (ATTR-CM) patient carrying a p.Ser43Asn mutation in the TTR gene.

Transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) is a life-threatening disease caused by the abnormal production of misfolded TTR protein by liver cells, which is then released systemically. Its amyloid deposition in the heart is linked to cardiac toxicity and progression toward heart failure. A human induced pluripotent stem cell (iPSC) line was generated from peripheral blood mononuclear cells (PBMCs) from a patient suffering familial transthyretin amyloid cardiomyopathy carrying a c.128G>A (p.Ser43Asn) mutation in the TTR gene. This iPSC line offers a useful resource to study the disease pathophysiology and a cell-based model for therapeutic discovery.

Loss of the matrix metalloproteinase-10 causes premature features of aging in satellite cells.

Aged muscles accumulate satellite cells with a striking decline response to damage. Although intrinsic defects in satellite cells themselves are the major contributors to aging-associated stem cell dysfunction, increasing evidence suggests that changes in the muscle-stem cell local microenvironment also contribute to aging. Here, we demonstrate that loss of the matrix metalloproteinase-10 (MMP-10) in young mice alters the composition of the muscle extracellular matrix (ECM), and specifically disrupts the extracellular matrix of the satellite cell niche. This situation causes premature features of aging in the satellite cells, contributing to their functional decline and a predisposition to enter senescence under proliferative pressure. Similarly, reduction of MMP-10 levels in young satellite cells from wild type animals induces a senescence response, while addition of the protease delays this program. Significantly, the effect of MMP-10 on satellite cell aging can be extended to another context of muscle wasting, muscular dystrophy. Systemic treatment of mdx dystrophic mice with MMP-10 prevents the muscle deterioration phenotype and reduces cellular damage in the satellite cells, which are normally under replicative pressure. Most importantly, MMP-10 conserves its protective effect in the satellite cell-derived myoblasts isolated from a Duchenne muscular dystrophy patient by decreasing the accumulation of damaged DNA. Hence, MMP-10 provides a previously unrecognized therapeutic opportunity to delay satellite cell aging and overcome satellite cell dysfunction in dystrophic muscles.

Revealing cell populations catching the early stages of human embryo development in naive pluripotent stem cell cultures.

Naive human pluripotent stem cells (hPSCs) are defined as the in vitro counterpart of the human preimplantation embryo's epiblast and are used as a model system to study developmental processes. In this study, we report the discovery and characterization of distinct cell populations coexisting with epiblast-like cells in 5iLAF naive human induced PSC (hiPSC) cultures. It is noteworthy that these populations closely resemble different cell types of the human embryo at early developmental stages. While epiblast-like cells represent the main cell population, interestingly we detect a cell population with gene and transposable element expression profile closely resembling the totipotent eight-cell (8C)-stage human embryo, and three cell populations analogous to trophectoderm cells at different stages of their maturation process: transition, early, and mature stages. Moreover, we reveal the presence of cells resembling primitive endoderm. Thus, 5iLAF naive hiPSC cultures provide an excellent opportunity to model the earliest events of human embryogenesis, from the 8C stage to the peri-implantation period.

One-Step In Vitro Generation of ETV2-Null Pig Embryos.

Each year, tens of thousands of people worldwide die of end-stage organ failure due to the limited availability of organs for use in transplantation. To meet this clinical demand, one of the last frontiers of regenerative medicine is the generation of humanized organs in pigs from pluripotent stem cells (PSCs) via blastocyst complementation. For this, organ-disabled pig models are needed. As endothelial cells (ECs) play a critical role in xenotransplantation rejection in every organ, we aimed to produce hematoendothelial-disabled pig embryos targeting the master transcription factor ETV2 via CRISPR-Cas9-mediated genome modification. In this study, we designed five different guide RNAs (gRNAs) against the DNA-binding domain of the porcine ETV2 gene, which were tested on porcine fibroblasts in vitro. Four out of five guides showed cleavage capacity and, subsequently, these four guides were microinjected individually as ribonucleoprotein complexes (RNPs) into one-cell-stage porcine embryos. Next, we combined the two gRNAs that showed the highest targeting efficiency and microinjected them at higher concentrations. Under these conditions, we significantly improved the rate of biallelic mutation. Hence, here, we describe an efficient one-step method for the generation of hematoendothelial-disabled pig embryos via CRISPR-Cas9 microinjection in zygotes. This model could be used in experimentation related to the in vivo generation of humanized organs.

Local Preirradiation of Infarcted Cardiac Tissue Substantially Enhances Cell Engraftment.

The success of cell therapy for the treatment of myocardial infarction depends on finding novel approaches that can substantially implement the engraftment of the transplanted cells. In order to enhance cell engraftment, most studies have focused on the pretreatment of transplantable cells. Here we have considered an alternative approach that involves the preconditioning of infarcted heart tissue to reduce endogenous cell activity and thus provide an advantage to our exogenous cells. This treatment is routinely used in other tissues such as bone marrow and skeletal muscle to improve cell engraftment, but it has never been taken in cardiac tissue. To avoid long-term cardiotoxicity induced by full heart irradiation we developed a rat model of a catheter-based heart irradiation system to locally impact a delimited region of the infarcted cardiac tissue. As proof of concept, we transferred ZsGreen+ iPSCs in the infarcted heart, due to their ease of use and detection. We found a very significant increase in cell engraftment in preirradiated rats. In this study, we demonstrate for the first time that preconditioning the infarcted cardiac tissue with local irradiation can substantially enhance cell engraftment.

Generation of NKX2.5GFP Reporter Human iPSCs and Differentiation Into Functional Cardiac Fibroblasts.

Direct cardiac reprogramming has emerged as an interesting approach for the treatment and regeneration of damaged hearts through the direct conversion of fibroblasts into cardiomyocytes or cardiovascular progenitors. However, in studies with human cells, the lack of reporter fibroblasts has hindered the screening of factors and consequently, the development of robust direct cardiac reprogramming protocols.In this study, we have generated functional human NKX2.5GFP reporter cardiac fibroblasts. We first established a new NKX2.5GFP reporter human induced pluripotent stem cell (hiPSC) line using a CRISPR-Cas9-based knock-in approach in order to preserve function which could alter the biology of the cells. The reporter was found to faithfully track NKX2.5 expressing cells in differentiated NKX2.5GFP hiPSC and the potential of NKX2.5-GFP + cells to give rise to the expected cardiac lineages, including functional ventricular- and atrial-like cardiomyocytes, was demonstrated. Then NKX2.5GFP cardiac fibroblasts were obtained through directed differentiation, and these showed typical fibroblast-like morphology, a specific marker expression profile and, more importantly, functionality similar to patient-derived cardiac fibroblasts. The advantage of using this approach is that it offers an unlimited supply of cellular models for research in cardiac reprogramming, and since NKX2.5 is expressed not only in cardiomyocytes but also in cardiovascular precursors, the detection of both induced cell types would be possible. These reporter lines will be useful tools for human direct cardiac reprogramming research and progress in this field.

Generation of four Isl1 reporter iPSC lines from cardiac and tail-tip fibroblasts derived from Ai6IslCre mouse.

Islet-1 (Isl1) is a transcription factor essential for life expressed in specific cells with different developmental origins. We have generated iPSC lines from fibroblasts of the transgenic Ai6 x Isl1-Cre (Ai6IslCre) mouse. Here we describe the complete characterization of four iPSC lines: ATCi-Ai6IslCre10, ATCi-Ai6IslCre35, ATCi-Ai6IslCre74 and ATCi-Ai6IslCre80.

Multipotent Adult Progenitor Cells Support Lymphatic Regeneration at Multiple Anatomical Levels during Wound Healing and Lymphedema.

Lymphatic capillary growth is an integral part of wound healing, yet, the combined effectiveness of stem/progenitor cells on lymphatic and blood vascular regeneration in wounds needs further exploration. Stem/progenitor cell transplantation also emerged as an approach to cure lymphedema, a condition caused by lymphatic system deficiency. While lymphedema treatment requires lymphatic system restoration from the capillary to the collector level, it remains undetermined whether stem/progenitor cells support a complex regenerative response across the entire anatomical spectrum of the system. Here, we demonstrate that, although multipotent adult progenitor cells (MAPCs) showed potential to differentiate down the lymphatic endothelial lineage, they mainly trophically supported lymphatic endothelial cell behaviour in vitro. In vivo, MAPC transplantation supported blood vessel and lymphatic capillary growth in wounds and restored lymph drainage across skin flaps by stimulating capillary and pre-collector vessel regeneration. Finally, human MAPCs mediated survival and functional reconnection of transplanted lymph nodes to the host lymphatic network by improving their (lymph)vascular supply and restoring collector vessels. Thus, MAPC transplantation represents a promising remedy for lymphatic system restoration at different anatomical levels and hence an appealing treatment for lymphedema. Furthermore, its combined efficacy on lymphatic and blood vascular growth is an important asset for wound healing.

Generation of Macaca fascicularis iPS cell line ATCi-MF1 from adult skin fibroblasts using non-integrative Sendai viruses.

We generated ATCi-MF1 induced pluripotent stem (iPS) cell line from Macaca fascicularis adult skin fibroblasts using non-integrative Sendai viruses carrying OCT3/4, KLF4, SOX2 and c-MYC. Once established, ATCi-MF1 cells present a normal karyotype, are Sendai virus-free and express pluripotency associated markers. Microsatellite markers analysis confirmed the origin of the iPS cells from the parental fibroblasts. Pluripotency was tested with the in vivo teratoma formation assay. ATCi-MF1 cell line may be a useful primate iPS cell model to test different experimental conditions where the use of human cells can imply ethical issues, as microinjection of pluripotent stem cells in pre-implantational embryos.

Isolation and characterization of Sprague-Dawley and Wistar Kyoto GFP rat embryonic stem cells.

We generated two rat embryonic stem cell (ESC) lines: ATCe-SD7.8 from Sprague-Dawley strain and ATCe-WK1 from Wistar Kyoto strain. Cells were marked with enhanced green fluorescent protein (eGFP) by transduction with a lentiviral vector. Cells present a normal karyotype and express pluripotency-associated markers. Pluripotency was tested in vivo with the teratoma formation assay. Cells maintain eGFP expression upon differentiation to the three-germ layers. These cells can be a useful tool for cell therapy studies and chimera generation as they can be easily tracked by eGFP expression.

Generation of a Sprague-Dawley-GFP rat iPS cell line.

We generated a rat iPSC line called ATCi-rSD95 from transgenic Sprague-Dawley GFP fetal fibroblasts. Established ATCi-rSD95 cells present a normal karyotype, silencing of the transgenes and express pluripotency-associated markers. Additionally, ATCi-rSD95 cells are able to form teratoma with differentiated cells derived from the three germ-layers that maintain the GFP expression.

Coronary risk in relation to genetic variation in MEOX2 and TCF15 in a Flemish population.

BACKGROUND: In mice MEOX2/TCF15 heterodimers are highly expressed in heart endothelial cells and are involved in the transcriptional regulation of lipid transport. In a general population, we investigated whether genetic variation in these genes predicted coronary heart disease (CHD). RESULTS: In 2027 participants randomly recruited from a Flemish population (51.0 % women; mean age 43.6 years), we genotyped six SNPs in MEOX2 and four in TCF15. Over 15.2 years (median), CHD, myocardial infarction, coronary revascularisation and ischaemic cardiomyopathy occurred in 106, 53, 78 and 22 participants. For SNPs, we contrasted CHD risk in minor-allele heterozygotes and homozygotes (variant) vs. major-allele homozygotes (reference) and for haplotypes carriers (variant) vs. non-carriers. In multivariable-adjusted analyses with correction for multiple testing, CHD risk was associated with MEOX2 SNPs (P ≤ 0.049), but not with TCF15 SNPs (P ≥ 0.29). The MEOX2 GTCCGC haplotype (frequency 16.5 %) was associated with the sex- and age-standardised CHD incidence (5.26 vs. 3.03 events per 1000 person-years; P = 0.036); the multivariable-adjusted hazard ratio [HR] of CHD was 1.78 (95 % confidence interval, 1.25-2.56; P = 0.0054). For myocardial infarction, coronary revascularisation, and ischaemic cardiomyopathy, the corresponding HRs were 1.96 (1.16-3.31), 1.87 (1.20-2.91) and 3.16 (1.41-7.09), respectively. The MEOX2 GTCCGC haplotype significantly improved the prediction of CHD over and beyond traditional risk factors and was associated with similar population-attributable risk as smoking (18.7 % vs. 16.2 %). CONCLUSIONS: Genetic variation in MEOX2, but not TCF15, is a strong predictor of CHD. Further experimental studies should elucidate the underlying molecular mechanisms.

Endothelial Msx1 transduces hemodynamic changes into an arteriogenic remodeling response.

Collateral remodeling is critical for blood flow restoration in peripheral arterial disease and is triggered by increasing fluid shear stress in preexisting collateral arteries. So far, no arterial-specific mediators of this mechanotransduction response have been identified. We show that muscle segment homeobox 1 (MSX1) acts exclusively in collateral arterial endothelium to transduce the extrinsic shear stimulus into an arteriogenic remodeling response. MSX1 was specifically up-regulated in remodeling collateral arteries. MSX1 induction in collateral endothelial cells (ECs) was shear stress driven and downstream of canonical bone morphogenetic protein-SMAD signaling. Flow recovery and collateral remodeling were significantly blunted in EC-specific Msx1/2 knockout mice. Mechanistically, MSX1 linked the arterial shear stimulus to arteriogenic remodeling by activating the endothelial but not medial layer to a proinflammatory state because EC but not smooth muscle cellMsx1/2 knockout mice had reduced leukocyte recruitment to remodeling collateral arteries. This reduced leukocyte infiltration in EC Msx1/2 knockout mice originated from decreased levels of intercellular adhesion molecule 1 (ICAM1)/vascular cell adhesion molecule 1 (VCAM1), whose expression was also in vitro driven by promoter binding of MSX1.

Meox2/Tcf15 heterodimers program the heart capillary endothelium for cardiac fatty acid uptake.

BACKGROUND: Microvascular endothelium in different organs is specialized to fulfill the particular needs of parenchymal cells. However, specific information about heart capillary endothelial cells (ECs) is lacking. METHODS AND RESULTS: Using microarray profiling on freshly isolated ECs from heart, brain, and liver, we revealed a genetic signature for microvascular heart ECs and identified Meox2/Tcf15 heterodimers as novel transcriptional determinants. This signature was largely shared with skeletal muscle and adipose tissue endothelium and was enriched in genes encoding fatty acid (FA) transport-related proteins. Using gain- and loss-of-function approaches, we showed that Meox2/Tcf15 mediate FA uptake in heart ECs, in part, by driving endothelial CD36 and lipoprotein lipase expression and facilitate FA transport across heart ECs. Combined Meox2 and Tcf15 haplodeficiency impaired FA uptake in heart ECs and reduced FA transfer to cardiomyocytes. In the long term, this combined haplodeficiency resulted in impaired cardiac contractility. CONCLUSIONS: Our findings highlight a regulatory role for ECs in FA transfer to the heart parenchyma and unveil 2 of its intrinsic regulators. Our insights could be used to develop new strategies based on endothelial Meox2/Tcf15 targeting to modulate FA transfer to the heart and remedy cardiac dysfunction resulting from altered energy substrate usage.

Infiltration of plasma rich in growth factors enhances in vivo angiogenesis and improves reperfusion and tissue remodeling after severe hind limb ischemia.

PRGF is a platelet concentrate within a plasma suspension that forms an in situ-generated fibrin-matrix delivery system, releasing multiple growth factors and other bioactive molecules that play key roles in tissue regeneration. This study was aimed at exploring the angiogenic and myogenic effects of PRGF on in vitro endothelial cells (HUVEC) and skeletal myoblasts (hSkMb) as well as on in vivo mouse subcutaneously implanted matrigel and on limb muscles after a severe ischemia. Human PRGF was prepared and characterized. Both proliferative and anti-apoptotic responses to PRGF were assessed in vitro in HUVEC and hSkMb. In vivo murine matrigel plug assay was conducted to determine the angiogenic capacity of PRGF, whereas in vivo ischemic hind limb model was carried out to demonstrate PRGF-driven vascular and myogenic regeneration. Primary HUVEC and hSkMb incubated with PRGF showed a dose dependent proliferative and anti-apoptotic effect and the PRGF matrigel plugs triggered an early and significant sustained angiogenesis compared with the control group. Moreover, mice treated with PRGF intramuscular infiltrations displayed a substantial reperfusion enhancement at day 28 associated with a fibrotic tissue reduction. These findings suggest that PRGF-induced angiogenesis is functionally effective at expanding the perfusion capacity of the new vasculature and attenuating the endogenous tissue fibrosis after a severe-induced skeletal muscle ischemia.

Unraveling a novel transcription factor code determining the human arterial-specific endothelial cell signature.

Endothelial cells (ECs) lining arteries and veins have distinct molecular/functional signatures. The underlying regulatory mechanisms are incompletely understood. Here, we established a specific fingerprint of freshly isolated arterial and venous ECs from human umbilical cord comprising 64 arterial and 12 venous genes, representing distinct functions/pathways. Among the arterial genes were 8 transcription factors (TFs), including Notch target HEY2, the current "gold standard" determinant for arterial EC (aEC) specification. Culture abrogated differential gene expression in part due to gradual loss of canonical Notch activity and HEY2 expression. Notably, restoring HEY2 expression or Delta-like4-induced Notch signaling in cultured ECs only partially reinstated the aEC gene signature, whereas combined overexpression of the 8 TFs restored this fingerprint more robustly. Whereas some TFs stimulated few genes, others boosted a large proportion of arterial genes. Although there was some overlap and cross-regulation, the TFs largely complemented each other in regulating the aEC gene profile. Finally, overexpression of the 8 TFs in human umbilical vein ECs conveyed an arterial-like behavior upon their implantation in a Matrigel plug in vivo. Thus, our study shows that Notch signaling determines only part of the aEC signature and identifies additional novel and complementary transcriptional players in the complex regulation of human arteriovenous EC identity.

COUP-TFII orchestrates venous and lymphatic endothelial identity by homo- or hetero-dimerisation with PROX1.

Endothelial cell (EC) identity is in part genetically predetermined. Transcription factor NR2F2 (also known as chicken ovalbumin upstream promoter transcription factor II, COUP-TFII) plays a key role in EC fate decision making; however, many of the underlying mechanisms remain enigmatic. In the present study, we demonstrate that NR2F2 differentially regulates gene expression of venous versus lymphatic ECs (LECs) and document a novel paradigm whereby NR2F2 homodimers induce a venous EC fate, while heterodimers with the LEC-specific transcription factor PROX1 instruct LEC lineage specification. NR2F2 homodimers inhibit arterial differentiation in venous ECs through direct binding to the promoter regions of the Notch target genes HEY1 and HEY2 (HEY1/2), whereas NR2F2/PROX1 heterodimers lack this inhibitory effect, resulting at least in part in non-canonical HEY1/2 expression in LECs. Furthermore, NR2F2/PROX1 heterodimers actively induce or are permissive for the expression of a major subset of LEC-specific genes. In addition to NR2F2/PROX1 heterodimerisation, the expression of HEY1 and some of these LEC-specific genes is dependent on PROX1 DNA binding. Thus, NR2F2 homodimers in venous ECs and NR2F2/PROX1 heterodimers in LECs differentially regulate EC subtype-specific genes and pathways, most prominently the Notch target genes HEY1/2. This novel mechanistic insight could pave the way for new therapeutic interventions for vascular-bed-specific disorders.

Quantification of miRNA-mRNA interactions.

miRNAs are small RNA molecules (' 22nt) that interact with their corresponding target mRNAs inhibiting the translation of the mRNA into proteins and cleaving the target mRNA. This second effect diminishes the overall expression of the target mRNA. Several miRNA-mRNA relationship databases have been deployed, most of them based on sequence complementarities. However, the number of false positives in these databases is large and they do not overlap completely. Recently, it has been proposed to combine expression measurement from both miRNA and mRNA and sequence based predictions to achieve more accurate relationships. In our work, we use LASSO regression with non-positive constraints to integrate both sources of information. LASSO enforces the sparseness of the solution and the non-positive constraints restrict the search of miRNA targets to those with down-regulation effects on the mRNA expression. We named this method TaLasso (miRNA-Target LASSO).We used TaLasso on two public datasets that have paired expression levels of human miRNAs and mRNAs. The top ranked interactions recovered by TaLasso are especially enriched (more than using any other algorithm) in experimentally validated targets. The functions of the genes with mRNA transcripts in the top-ranked interactions are meaningful. This is not the case using other algorithms.TaLasso is available as Matlab or R code. There is also a web-based tool for human miRNAs at http://talasso.cnb.csic.es/.

Transcription factor COUP-TFII is indispensable for venous and lymphatic development in zebrafish and Xenopus laevis.

Transcription factors play a central role in cell fate determination. Gene targeting in mice revealed that Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII, also known as Nuclear Receptor 2F2 or NR2F2) induces a venous phenotype in endothelial cells (ECs). More recently, NR2F2 was shown to be required for initiating the expression of Prox1, responsible for lymphatic commitment of venous ECs. Small animal models like zebrafish embryos and Xenopus laevis tadpoles have been very useful to elucidate mechanisms of (lymph) vascular development. Therefore, the role of NR2F2 in (lymph) vascular development was studied by eliminating its expression in these models. Like in mice, absence of NR2F2 in zebrafish resulted in distinct vascular defects including loss of venous marker expression, major trunk vessel fusion and vascular leakage. Both in zebrafish and Xenopus the development of the main lymphatic structures was severely hampered. NR2F2 knockdown significantly decreased prox1 expression in zebrafish ECs and the same manipulation affected lymphatic (L)EC commitment, migration and function in Xenopus tadpoles. Therefore, the role of NR2F2 in EC fate determination is evolutionary conserved.