Dries Feyen

A Fast pH‐Switchable and Self‐Healing Supramolecular Hydrogel Carrier for Guided, Local Catheter Injection in the Infarcted Myocardium

Minimally invasive intervention strategies after myocardial infarction use state-of-the-art catheter systems that are able to combine mapping of the infarcted area with precise, local injection of drugs. To this end, catheter delivery of drugs that are not immediately pumped out of the heart is still challenging, and requires a carrier matrix that in the solution state can be injected through a long catheter, and instantaneously gelates at the site of injection. To address this unmet need, a pH-switchable supramolecular hydrogel is developed. The supramolecular hydrogel is switched into a liquid at pH > 8.5, with a viscosity low enough to enable passage through a 1-m long catheter while rapidly forming a hydrogel in contact with tissue. The hydrogel has self-healing properties taking care of adjustment to the injection site. Growth factors are delivered from the hydrogel thereby clearly showing a reduction of infarct scar in a pig myocardial infarction model.

Human versus porcine mesenchymal stromal cells: phenotype, differentiation potential, immunomodulation and cardiac improvement after transplantation

Although mesenchymal stromal cells (MSCs) have been applied clinically to treat cardiac diseases, it is unclear how and to which extent transplanted MSCs exert their beneficial effects. To address these questions, pre‐clinical MSC administrations are needed for which pigs appear to be the species of choice. This requires the use of porcine cells to prevent immune rejection. However, it is currently unknown to what extent porcine MSCs (pMSCs) resemble human MSCs (hMSCs). Aim of this study was to compare MSC from porcine bone marrow (BM) with human cells for phenotype, multi‐lineage differentiation potential, immune‐modulatory capacity and the effect on cardiac function after transplantation in a mouse model of myocardial infarction. Flow cytometric analysis revealed that pMSC expressed surface antigens also found on hMSC, including CD90, MSCA‐1 (TNAP/W8B2 antigen), CD44, CD29 and SLA class I. Clonogenic outgrowth was significantly enriched following selection of CD271+ cells from BM of human and pig (129 ± 29 and 1961 ± 485 fold, respectively). hMSC and pMSC differentiated comparably into the adipogenic, osteogenic or chondrogenic lineages, although pMSC formed fat much faster than hMSC. Immuno‐modulation, an important feature of hMSC, was clearly demonstrated for pMSC when co‐cultured with porcine peripheral blood cells stimulated with PMA and pIL‐2. Finally, pMSC transplantation after myocardial infarction attenuated adverse remodelling to a similar extent as hMSC when compared to control saline injection. These findings demonstrate that pMSCs have comparable characteristics and functionality with hMSCs, making reliable extrapolation of pre‐clinical pMSC studies into a clinical setting very well possible.

Different types of cultured human adult cardiac progenitor cells have a high degree of transcriptome similarity.

The discovery and isolation of different resident cardiac progenitor cells (CPCs) a decade ago, as described by several research groups, stimulated the use of these cells for cardiac regeneration. Human CPCs are moving towards the clinic as one of the most promising cell types for cardiac repair, but the extent to which their molecular profiles vary as a result of donor heterogeneity or different isolation methods remain unclear. Defining a common molecular profile that defines CPC’s is therefore an important goal. Similarly, identifying robust and multilaboratory isolation and culture protocols that generate reproducible cell populations from genetically diverse donors is critical for their translational success. In this respect, we collected human auricle biopsy samples anonymously from 20 different adult patients that underwent bypass surgery and generated a total number of 33 different cardiac derived progenitor cell (CPC) lines (Table S1). Human CPCs were isolated according the original published protocol, based on c-kit 1 or Sca-1 2 expression or auricles were cut in 1 mm3 parts and cultured as explants to obtain Cardiospheres (CSps) 3 and Cardiosphere Derived Cells (CDCs) 4. CPCs were subsequently propagated in a panel of different media formulations, either in their originally described culture media or switched to media and culture coatings of the other CPC subsets (Figure S1, Table S7). When comparing individual CPC cell-lines, isolated with different methodologies, they shared a high degree of similarities and correlation in gene expression patterns (Fig. ​(Fig.1B).1B). By averaging expression profiles of individual CPC conditions, thereby reducing donor variability, similarities increased even more, ranging from 0.92 to 0.96 (Fig. ​(Fig.1C;1C; Table S2). These results suggest that individual donor differences were larger than influences of isolation and medium conditions. Moreover, the strongest correlations between the different CPC lines were observed when cells were isolated and cultured in the same conditions. Among the different CPCs, spheres-growing CSps showed the least correlation (0.91–0.96), while monolayer-growing CPCs shared higher correlations among them (0.96–0.98). We performed a moderated t-test to evaluate significant differentially expressed genes between the individual samples (Tables S3 and S4). Out of the 13,073 analysd genes, we found only few genes differentially expressed in 5 of 20 different monolayer-cultured CPC cell-lines comparisons. Only when the 3D-cultured CSps were compared with the other CPCs more differently expressed genes could be identified. Although only limited genes were different, we further explored if we could identify differences in gene patterns between the different CPC populations, based on selected genes important for stem cell-maintenance, their growth and biology. In particular, we evaluated genes involved in the regulation of different stem cell pathways like TGF-β, Wnt, NFkB, p53, JAK/STAT, Notch and Hedgehog (Fig. S2A), cell cycle (Fig. S2B), stem cell transcription factors (Fig. S2C), and growth factors, cytokines and chemokines (Fig. S2D). Detailed heat map analysis showed again; however, a very similar profile among all samples, with small differences mainly related to individual donors and not to different cell types or conditions (Fig. S2). Since CSps and monolayer growing CPCs have differently expressed patterns, we selected all the significantly differentially expressed genes that displayed a two fold or more difference and compared them with CDCs, and c-Kit and Sca-1+ CPCs monolayer-cultures (Table S6). Ingenuity pathway analysis identified a gene network in CSps that is enriched in genes encoding for growth factor production and signalling molecules involved in the development of cardiac muscle, vasculogenesis and angiogenesis (Fig. ​(Fig.2).2). Among them BMP-2, HGF, LIF, PTGS-2, VEGFA and PDGFRB are known to play an important role during cardiac development. Moreover, having a protective effect on a developing heart failure. Figure 1 Experimental design of the project (A) and hierarchical clustering of CPCs samples (B and C). Sca-1+ cells were isolated from human auricle biopsy and cultured in gelatin coated flask and Sca-1 medium (Sca GEL S-MED) (2) (d). After expansion cells were ... Figure 2 Ingenuity molecular networks analysis of the differentially expressed genes. Fold difference ≥2; p<0.05. (A) Differentially regulated genes between CSps and Sca GEL SP++ in Cardiovascular System Development and Function, Embryonic Development, ... Taken together, our data suggest that human CPCs can be isolated from patient heart biopsies using different markers, such as c-kit or Sca-1- like, and alternative methodologies, via direct cell isolation or via explant culture, such as CSps and CDCs. For the first time, however, we showed that upon culture expansion, these cell populations have a very similar gene expression profile, even more pronounced when cultured in comparable culture conditions and even transcended by donor differences. Among the different CPCs analysed, CSps are the most different, probably because of the unselected cell populations and containing more supporting cell population that form CSps and their particular 3D culture structure and thereby different interactions and growing conditions. Surprisingly CDCs, which is a cell population derived from CSps, are more similar with other antigen selected CPCs rather than with CSps, confirming the idea that monolayer and high proliferative culture condition might play an important role in minimizing the differences among the different CPCs analysed. Recently, Dey et al. isolated murine CPCs, based on different surface markers 5, and showed that these, non-cultured cells, represent progenitor cell populations at different stages of cardiac commitment 5. In our study, we did not observe such differences between the different human monolayer CPCs population upon culture propagation. A similar stage difference, however, might be present in situ in humans as well but lost upon culture expansion. The expression of these different stem cell markers and their co-expression probably represent different developmental and/or physiological stages of CPCs, rather than intrinsic different CPC populations. For future translation for cardiac cell therapy, our results suggest that we need to take into account the cell donor variability between patients more than the isolation methodology, and further study the correlation between CPC characteristics and e.g. the diseased status of a patient. Our findings are of fundamental importance to create a consensus among different scientists in the field of myocardial regeneration, which should help align future clinical approaches to improve the reported beneficial effects of cell therapy for heart disease by using cardiac derived progenitor cell populations.

DNA Nuclear Targeting Sequences for Non-Viral Gene Delivery

ABSTRACTPurposeTo evaluate if introduction of DNA nuclear Targeting Sequences (DTS; i.e. recognition sequences for endogenous DNA-binding proteins) in plasmid DNA (pDNA) leads to increased transfection efficiency of non-viral gene delivery by virtue of enhanced nuclear import of the pDNA.MethodsA set of DTS was identified and cloned into EGFP-reporter plasmids controlled by the CMV-promoter. These pDNA constructs were delivered into A431 and HeLa cells using standard electroporation, pEI-based polyfection or lipofection methods. The amount of pDNA delivered into the nucleus was determined by qPCR; transfection efficiency was determined by flow cytometry.ResultsNeither of these DTS increased transgene expression. We varied several parameters (mitotic activity, applied dose and delivery strategy), but without effect. Although upregulated transgene expression was observed after stimulation with TNF-α, this effect could be ascribed to non-specific upregulation of transcription rather than enhanced nuclear import. Nuclear copy numbers of plasmids containing or lacking a DTS did not differ significantly after lipofectamine-based transfection in dividing and non-dividing cells.ConclusionNo beneficial effects of DTS on gene expression or nuclear uptake were observed in this study.

Intramyocardial stem cell injection: go(ne) with the flow.

In this study, we visualize the real-time dynamics of intramyocardial stem-cell injections. This shows a massive, immediate wash-out via venous drainage, accounting for the low retention. The use of carriers reduces this outflow.

Increasing short-term cardiomyocyte progenitor cell (CMPC) survival by necrostatin-1 did not further preserve cardiac function

AIMS One of the main limitations for an effective cell therapy for the heart is the poor cell engraftment after implantation, which is partly due to a large percentage of cell death in the hostile myocardium. In the present study, we investigated the utilization of necrostatin-1 (Nec-1) as a possible attenuator of cell death in cardiomyocyte progenitor cells (CMPCs). METHODS AND RESULTS In a mouse model of myocardial infarction, survival of CMPCs 3 days after intra-myocardial injection was 39 ± 9% higher in cells pretreated with the Nec-1 compound. However, the increase in cell number was not sustained over 28 days, and did not translate into improved cardiac function (ejection fraction %, 20.6 ± 2.1 vs. 21.4 ± 2.5 for vehicle and Nec-1-treated CMPC, respectively). Nonetheless, Nec-1 rescued CMPCs remained functionally competent. CONCLUSION A pharmacological pretreatment approach to solely enhance cell survival on the short term does not seem to be effective strategy to improve cardiac cell therapy with CMPCs.

Melt electrospinning writing of poly-Hydroxymethylglycolide-co-ε-Caprolactone-based scaffolds for cardiac tissue engineering

Current limitations in cardiac tissue engineering revolve around the inability to fully recapitulate the structural organization and mechanical environment of native cardiac tissue. This study aims at developing organized ultrafine fiber scaffolds with improved biocompatibility and architecture in comparison to the traditional fiber scaffolds obtained by solution electrospinning. This is achieved by combining the additive manufacturing of a hydroxyl-functionalized polyester, (poly(hydroxymethylglycolide-co-ε-caprolactone) (pHMGCL), with melt electrospinning writing (MEW). The use of pHMGCL with MEW vastly improves the cellular response to the mechanical anisotropy. Cardiac progenitor cells (CPCs) are able to align more efficiently along the preferential direction of the melt electrospun pHMGCL fiber scaffolds in comparison to electrospun poly(ε-caprolactone)-based scaffolds. Overall, this study describes for the first time that highly ordered microfiber (4.0-7.0 µm) scaffolds based on pHMGCL can be reproducibly generated with MEW and that these scaffolds can support and guide the growth of CPCs and thereby potentially enhance their therapeutic potential.

Stem cell-based therapy: Improving myocardial cell delivery.

Stem cell-based therapies form an exciting new class of medicine that attempt to provide the body with the building blocks required for the reconstruction of damaged organs. However, delivering cells to the correct location, while preserving their integrity and functional properties, is a complex undertaking. These challenges have led to the development of a highly dynamic interdisciplinary research field, wherein medical, biological, and chemical sciences have collaborated to develop strategies to overcome the physiological barriers imposed on the cellular therapeutics. In this respect, improving the acute retention and subsequent survival of stem cells is key to effectively increase the effect of the therapy, while proper tissue integration is imperative for stem cells to functionally replace lost cells in damaged organs. In this review, we will use the heart as an example to highlight the current knowledge of therapeutic stem cell utilization, the existing pitfalls and limitations, and the approaches that have been developed to overcome them.

Gelatin Microspheres as Vehicle for Cardiac Progenitor Cells Delivery to the Myocardium

Inadequate cell retention and survival in cardiac stem cell therapy seems to be reducing the therapeutic effect of the injected stem cells. In order to ameliorate their regenerative effects, various biomaterials are being investigated for their potential supportive properties. Here, gelatin microspheres (MS) are utilized as microcarriers to improve the delivery and therapeutic efficacy of cardiac progenitor cells (CPCs) in the ischemic myocardium. The gelatin MS, generated from a water-in-oil emulsion, are able to accommodate the attachment of CPCs, thereby maintaining their cardiogenic potential. In a mouse model of myocardial infarction, we demonstrated the ability of these microcarriers to substantially enhance cell engraftment in the myocardium as indicated by bioluminescent imaging and histological analysis. However, despite an observed tenfold increase in CPC numbers in the myocardium, echocardiography, and histology reveals that mice treated with MS-CPCs show marginal improvement in cardiac function compared to CPCs only. Overall, a straightforward and translational approach is developed to increase the retention of stem cells in the ischemic myocardium. Even though the current biomaterial setup with CPCs as cell source does not translate into improved therapeutic action, coupling this developed technology with stem cell-derived cardiomyocytes can lead to an effective remuscularization therapy.

SCA1+ Cells from the Heart Possess a Molecular Circadian Clock and Display Circadian Oscillations in Cellular Functions

Summary Stem cell antigen 1-positive (SCA1+) cells (SPCs) have been investigated in cell-based cardiac repair and pharmacological research, although improved cardiac function after injection has been variable and the mode of action remains unclear. Circadian (24-hr) rhythms are biorhythms regulated by molecular clocks that play an important role in (patho)physiology. Here, we describe (1) the presence of a molecular circadian clock in SPCs and (2) circadian rhythmicity in SPC function. We isolated SPCs from human fetal heart and found that these cells possess a molecular clock based on typical oscillations in core clock components BMAL1 and CRY1. Functional analyses revealed that circadian rhythmicity also governs SPC proliferation, stress tolerance, and growth factor release, with large differences between peaks and troughs. We conclude that SPCs contain a circadian molecular clock that controls crucial cellular functions. Taking circadian rhythms into account may improve reproducibility and outcome of research and therapies using SPCs.