Head-to-head comparison of relevant cell sources of small extracellular vesicles for cardiac repair: Superiority of embryonic stem cells.

Small extracellular vesicles (sEV) derived from various cell sources have been demonstrated to enhance cardiac function in preclinical models of myocardial infarction (MI). The aim of this study was to compare different sources of sEV for cardiac repair and determine the most effective one, which nowadays remains limited. We comprehensively assessed the efficacy of sEV obtained from human primary bone marrow mesenchymal stromal cells (BM-MSC), human immortalized MSC (hTERT-MSC), human embryonic stem cells (ESC), ESC-derived cardiac progenitor cells (CPC), human ESC-derived cardiomyocytes (CM), and human primary ventricular cardiac fibroblasts (VCF), in in vitro models of cardiac repair. ESC-derived sEV (ESC-sEV) exhibited the best pro-angiogenic and anti-fibrotic effects in vitro. Then, we evaluated the functionality of the sEV with the most promising performances in vitro, in a murine model of MI-reperfusion injury (IRI) and analysed their RNA and protein compositions. In vivo, ESC-sEV provided the most favourable outcome after MI by reducing adverse cardiac remodelling through down-regulating fibrosis and increasing angiogenesis. Furthermore, transcriptomic, and proteomic characterizations of sEV derived from hTERT-MSC, ESC, and CPC revealed factors in ESC-sEV that potentially drove the observed functions. In conclusion, ESC-sEV holds great promise as a cell-free treatment for promoting cardiac repair following MI.

Lipid Nanoparticles Deliver the Therapeutic VEGFA mRNA In Vitro and In Vivo and Transform Extracellular Vesicles for Their Functional Extensions.

Lipid nanoparticles (LNPs) are currently used to transport functional mRNAs, such as COVID-19 mRNA vaccines. The delivery of angiogenic molecules, such as therapeutic VEGF-A mRNA, to ischemic tissues for producing new blood vessels is an emerging strategy for the treatment of cardiovascular diseases. Here, the authors deliver VEGF-A mRNA via LNPs and study stoichiometric quantification of their uptake kinetics and how the transport of exogenous LNP-mRNAs between cells is functionally extended by cells' own vehicles called extracellular vesicles (EVs). The results show that cellular uptake of LNPs and their mRNA molecules occurs quickly, and that the translation of exogenously delivered mRNA begins immediately. Following the VEGF-A mRNA delivery to cells via LNPs, a fraction of internalized VEGF-A mRNA is secreted via EVs. The overexpressed VEGF-A mRNA is detected in EVs secreted from three different cell types. Additionally, RNA-Seq analysis reveals that as cells' response to LNP-VEGF-A mRNA treatment, several overexpressed proangiogenic transcripts are packaged into EVs. EVs are further deployed to deliver VEGF-A mRNA in vitro and in vivo. Upon equal amount of VEGF-A mRNA delivery via three EV types or LNPs in vitro, EVs from cardiac progenitor cells are the most efficient in promoting angiogenesis per amount of VEGF-A protein produced. Intravenous administration of luciferase mRNA shows that EVs could distribute translatable mRNA to different organs with the highest amounts of luciferase detected in the liver. Direct injections of VEGF-A mRNA (via EVs or LNPs) into mice heart result in locally produced VEGF-A protein without spillover to liver and circulation. In addition, EVs from cardiac progenitor cells cause minimal production of inflammatory cytokines in cardiac tissue compared with all other treatment types. Collectively, the data demonstrate that LNPs transform EVs as functional extensions to distribute therapeutic mRNA between cells, where EVs deliver this mRNA differently than LNPs.

Pluripotent stem cells derived from mouse and human white mature adipocytes.

White mature adipocytes give rise to so-called dedifferentiated fat (DFAT) cells that spontaneously undergo multilineage differentiation. In this study, we defined stem cell characteristics of DFAT cells as they are generated from adipocytes and the relationship between these characteristics and lineage differentiation. Both mouse and human DFAT cells, prepared from adipose tissue and lipoaspirate, respectively, showed evidence of pluripotency, with a maximum 5-7 days after adipocyte isolation. The DFAT cells spontaneously formed clusters in culture, which transiently expressed multiple stem cell markers, including stage-specific embryonic antigens, and Sca-1 (mouse) and CD105 (human), as determined by real-time polymerase chain reaction, fluorescence-activated cell sorting, and immunostaining. As the stem cell markers decreased, markers characteristic of the three germ layers and specific lineage differentiation, such as α-fetoprotein (endoderm, hepatic), Neurofilament-66 (ectoderm, neurogenic), and Troponin I (mesoderm, cardiomyogenic), increased. However, no teratoma formation was detected after injection in immunodeficient mice. A novel modification of the adipocyte isolation aimed at ensuring the initial purity of the adipocytes and avoiding ceiling culture allowed isolation of DFAT cells with pluripotent characteristics. Thus, the adipocyte-derived DFAT cells represent a plastic stem cell population that is highly responsive to changes in culture conditions and may benefit cell-based therapies.

Endothelial differentiation in multipotent cells derived from mouse and human white mature adipocytes.

White mature adipocytes give rise to multipotent cells, so-called de-differentiated fat (DFAT) cells, when losing their fat in culture. The objective of this study was to examine the ability of DFAT cells to give rise to endothelial cells (ECs) in vitro and vivo. We demonstrate that mouse and human DFAT cells, derived from adipose tissue and lipospirate, respectively, initially lack expression of CD34, CD31, CD146, CD45 and pericyte markers, distinguishing them from progenitor cells previously identified in adipose stroma. The DFAT cells spontaneously differentiate into vascular ECs in vitro, as determined by real-time PCR, fluorescence activated cell sorting, immunostaining, and formation of tube structures. Treatment with bone morphogenetic protein (BMP)4 and BMP9, important in regulating angiogenesis, significantly enhances the EC differentiation. Furthermore, adipocyte-derived cells from Green Fluorescent Protein-transgenic mice were detected in the vasculature of infarcted myocardium up to 6 weeks after ligation of the left anterior descending artery in mice. We conclude that adipocyte-derived multipotent cells are able to spontaneously give rise to ECs, a process that is promoted by BMPs and may be important in cardiovascular regeneration and in physiological and pathological changes in fat and other tissues.

Reprogrammed mouse fibroblasts differentiate into cells of the cardiovascular and hematopoietic lineages.

Forced expression of the four transcription factors Oct4, Sox2, c-Myc, and Klf4 is sufficient to confer a pluripotent state upon the murine fibroblast genome, generating induced pluripotent stem (iPS) cells. Although the differentiation potential of these cells is thought to be equivalent to that of embryonic stem (ES) cells, it has not been rigorously determined. In this study, we sought to identify the capacity of iPS cells to differentiate into Flk1-positive progenitors and their mesodermal progeny, including cells of the cardiovascular and hematopoietic lineages. Immunostaining of tissues from iPS cell-derived chimeric mice demonstrated that iPS cells could contribute in vivo to cardiomyocytes, smooth muscle cells, endothelial cells, and hematopoietic cells. To compare the in vitro differentiation potential of murine ES and iPS cells, we either induced embryoid body (EB) formation of each cell type or cultured the cells on collagen type IV (ColIV), an extracellular matrix protein that had been reported to direct murine ES cell differentiation to mesodermal lineages. EB formation and exposure to ColIV both induced iPS cell differentiation into cells that expressed cardiovascular and hematopoietic markers. To determine whether ColIV-differentiated iPS cells contained a progenitor cell with cardiovascular and hematopoietic differentiation potential, Flk1-positive cells were isolated by magnetic cell sorting and exposed to specific differentiation conditions, which induced differentiation into functional cardiomyocytes, smooth muscle cells, endothelial cells, and hematopoietic cells. Our data demonstrate that murine iPS cells, like ES cells, can differentiate into cells of the cardiovascular and hematopoietic lineages and therefore may represent a valuable cell source for applications in regenerative medicine. Disclosure of potential conflicts of interest is found at the end of this article.

The role of cytoprotective cytokines in cardiac ischemia/reperfusion injury.

The mechanism(s) underlying the beneficial effects of adult mesenchymal stem cells (MSCs) after myocardial infarction (MI) is poorly understood. One possible explanation is the ability of MSCs to secrete cytokines, which modulate cardiomyocyte survival and function. MSCs express at least two cytoprotective cytokines, hepatocyte growth factor (HGF) and stromal cell-derived factor-1 alpha (CXCL12). The aim of our study was to compare the effects of these two cytokines administered acutely post-MI. We subjected adult male Lewis rats to myocardial ischemia/reperfusion injury. Immediately upon reperfusion, polymers saturated with HGF or CXCL12 were placed onto the infarcted anterior wall and the rats were allowed to recover. Echocardiographic analysis at 4 wk post-MI to assess left ventricular (LV) function revealed that LV ejection fraction was increased in the HGF treated group compared with the phosphate-buffered saline (PBS) control group. Likewise, LV end diastolic dimension was reduced in the HGF treated group compared with the PBS control group. Similarly, invasive hemodynamics at 12 wk showed improved contractility and relaxation in the HGF treated group compared with the PBS control group. In contrast, no significant effect on LV function was seen in the CXCL12 treated group. To determine the potential mechanism for this effect, infarct size (IFS) at 72 h was determined. IFS was decreased 4.2-fold in the HGF treated group compared with the PBS control group. Thus, HGF acutely post-MI using polymer delivery reduces IFS, leading to beneficial effects on post-MI LV remodeling.

Optimized preservation of extracellular matrix in cardiac tissues: implications for long-term graft durability.

BACKGROUND: Cryopreservation of human tissues, particularly heart valves, is widespread in clinical practice although the effects of this process on underlying tissue structures and its potential impact on valve durability have been poorly studied. Multiphoton imaging and second-harmonic generation (SHG) microscopy permit high-resolution, noninvasive analysis of living tissues at a subcellular level. In the present study we used these novel imaging modalities to compare the effects of vitreous and frozen cryopreservation on the extracellular matrix (ECM) of cardiac tissues. METHODS: Conventional histology, electron microscopy, and multiphoton imaging to obtain autofluorescence and SHG images were performed on cardiac tissues to characterize the ECM in fresh, vitrified, and frozen cryopreserved tissues. RESULTS: Autofluorescence and particularly SHG images revealed that conventional frozen cryopreservation of cardiac valves, when compared with fresh or vitrified tissues, leads to the loss of normal ECM structures in valve leaflets. Similar results were found in all other cardiac tissues suggesting that structural deterioration of the ECM is a common consequence of frozen cryopreservation. CONCLUSIONS: Our results demonstrate that conventional cryopreservation, when compared with fresh or vitrified tissues, causes more destruction of normal ECM structure, which might contribute to eventual graft dysfunction. Whether vitrification preservation will translate into greater durability or less valve failure will need to be determined.

Collagen IV induces trophoectoderm differentiation of mouse embryonic stem cells.

The earliest segregation of lineages in the developing embryo is the commitment of cells to the inner cell mass or the trophoectoderm in preimplantation blastocysts. The exogenous signals that control commitment to a particular cell lineage are poorly understood; however, it has been suggested that extracellular "niche" and extracellular matrix, in particular, play an important role in determining the developmental fate of stem cells. Collagen IV (ColIV) has been reported to direct embryonic stem (ES) cell differentiation to mesodermal lineages in both mouse and human ES cells. To define the effects of ColIV on ES cell differentiation and to identify the resulting heterogeneous cell types, we performed microarray analyses and determined global gene expression. We observed that ColIV induced the expression of mesodermal genes specific to hematopoietic, endothelial, and smooth muscle cells and, surprisingly, also a panel of trophoectoderm-restricted markers. This effect was specific to collagen IV, as no trophoblast differentiation was seen on collagen I, laminin, or fibronectin. Stimulation with basic fibroblast growth factor (FGF) or FGF4 increased the number of trophoectodermal cells. These cells were isolated under clonal conditions and successfully differentiated into a variety of trophoblast derivatives. Interestingly, differentiation of ES cells to trophoblastic lineages was only seen in ES cell lines maintained on embryonic feeder layers and was caudal-type homeobox protein 2 (Cdx2)-dependent, consistent with Cdx2's postulated role in trophoectoderm commitment. Our data suggest that, given the appropriate extracellular stimuli, mouse embryonic stem cells can differentiate into trophoectoderm. Disclosure of potential conflicts of interest is found at the end of this article.

Co-culture of endothelial cells and smooth muscle cells affects gene expression of angiogenic factors.

Endothelial cells (EC) are in contact with the underlying smooth muscle cells (SMC). The interactions between EC and SMC in the vessel wall are considered to be involved in the control of growth and function of blood vessels. A co-culture system of EC and SMC and a method for separation of these cells was developed in order to investigate whether the presence of physical contact between EC and SMC affected the gene expression of angiogenic factors. Human EC and SMC were prepared from the great saphenous veins. Autologous EC were added on top of the confluent layer of SMC. After 72 h in co-culture, the EC were magnetically separated from SMC with the use of superparamagnetic beads. RT-PCR products for bFGF, bFGFR, VEGF, PDGF-AA, PDGF-BB, TGF-beta, and beta-actin were analyzed to study the mRNA expressions. The protein level of selected factors was studied by ELISA technique. In co-cultured SMC there was a statistically significant higher gene expression of VEGF, PDGF-AA, PDGF-BB, and TGF-beta and significant lower gene expression of bFGF and its receptor than in single cultured SMC. The protein level of PDGF-BB and TGF-beta was also significantly higher in co-cultured SMC. In co-cultured EC there were no significant differences in gene expression of PDGF-AA, PDGF-BB, and TGF-beta compared with single cultured EC. The gene expression and protein synthesis of VEGF was significantly higher in co-cultured EC. The findings from the present study suggest that cell-cell interactions of EC and SMC affect the gene and protein expression of angiogenic factors.