Development of an iPSC-derived tissue-resident macrophage-based platform for the in vitro immunocompatibility assessment of human tissue engineered matrices.

Upon implanting tissue-engineered heart valves (TEHVs), blood-derived macrophages are believed to orchestrate the remodeling process. They initiate the immune response and mediate the remodeling of the TEHV, essential for the valve's functionality. The exact role of another macrophage type, the tissue-resident macrophages (TRMs), has not been yet elucidated even though they maintain the homeostasis of native tissues. Here, we characterized the response of hTRM-like cells in contact with a human tissue engineered matrix (hTEM). HTEMs comprised intracellular peptides with potentially immunogenic properties in their ECM proteome. Human iPSC-derived macrophages (iMφs) could represent hTRM-like cells in vitro and circumvent the scarcity of human donor material. iMφs were derived and after stimulation they demonstrated polarization towards non-/inflammatory states. Next, they responded with increased IL-6/IL-1ß secretion in separate 3/7-day cultures with longer production-time-hTEMs. We demonstrated that iMφs are a potential model for TRM-like cells for the assessment of hTEM immunocompatibility. They adopt distinct pro- and anti-inflammatory phenotypes, and both IL-6 and IL-1ß secretion depends on hTEM composition. IL-6 provided the highest sensitivity to measure iMφs pro-inflammatory response. This platform could facilitate the in vitro immunocompatibility assessment of hTEMs and thereby showcase a potential way to achieve safer clinical translation of TEHVs.

Patch Materials for Pulmonary Artery Arterioplasty and Right Ventricular Outflow Tract Augmentation: A Review.

Patch augmentation of the right ventricular outflow tract (RVOT) and pulmonary artery (PA) arterioplasty are relatively common procedures in the surgical treatment of patients with congenital heart disease. To date, several patch materials have been applied with no agreed upon clinical standard. Each patch type has unique performance characteristics, cost, and availability. There are limited data describing the various advantages and disadvantages of different patch materials. We performed a review of studies describing the clinical performance of various RVOT and PA patch materials and found a limited but growing body of literature. Short-term clinical performance has been reported for a multitude of patch types, but comparisons are limited by inconsistent study design and scarce histologic data. Standard clinical criteria for assessment of patch efficacy and criteria for intervention need to be applied across patch types. The field is progressing with improvements in outcomes due to newer patch technologies focused on reducing antigenicity and promoting neotissue formation which may have the ability to grow, remodel, and repair.

Xeno-free induced pluripotent stem cell-derived neural progenitor cells for in vivo applications.

BACKGROUND: Currently, there is no regenerative therapy for patients with neurological and neurodegenerative disorders. Cell-therapies have emerged as a potential treatment for numerous brain diseases. Despite recent advances in stem cell technology, major concerns have been raised regarding the feasibility and safety of cell therapies for clinical applications. METHODS: We generated good manufacturing practice (GMP)-compatible neural progenitor cells (NPCs) from transgene- and xeno-free induced pluripotent stem cells (iPSCs) that can be smoothly adapted for clinical applications. NPCs were characterized in vitro for their differentiation potential and in vivo after transplantation into wild type as well as genetically immunosuppressed mice. RESULTS: Generated NPCs had a stable gene-expression over at least 15 passages and could be scaled for up to 1018 cells per initially seeded 106 cells. After withdrawal of growth factors in vitro, cells adapted a neural fate and mainly differentiated into active neurons. To ensure a pure NPC population for in vivo applications, we reduced the risk of iPSC contamination by applying micro RNA-switch technology as a safety checkpoint. Using lentiviral transduction with a fluorescent and bioluminescent dual-reporter construct, combined with non-invasive in vivo bioluminescent imaging, we longitudinally tracked the grafted cells in healthy wild-type and genetically immunosuppressed mice as well as in a mouse model of ischemic stroke. Long term in-depth characterization revealed that transplanted NPCs have the capability to survive and spontaneously differentiate into functional and mature neurons throughout a time course of a month, while no residual pluripotent cells were detectable. CONCLUSION: We describe the generation of transgene- and xeno-free NPCs. This simple differentiation protocol combined with the ability of in vivo cell tracking presents a valuable tool to develop safe and effective cell therapies for various brain injuries.

Heterogeneous expression of ACE2 and TMPRRS2 in mesenchymal stromal cells.

The outbreak of COVID-19 has become a serious public health emergency. The virus targets cells by binding the ACE2 receptor. After infection, the virus triggers in some humans an immune storm containing the release of proinflammatory cytokines and chemokines followed by multiple organ failure. Several vaccines are enrolled, but an effective treatment is still missing. Mesenchymal stem cells (MSCs) have shown to secrete immunomodulatory factors that suppress this cytokine storm. Therefore, MSCs have been suggested as a potential treatment option for COVID-19. We report here that the ACE2 expression is minimal or nonexistent in MSC derived from three different human tissue sources (adipose tissue, umbilical cord Wharton`s jelly and bone marrow). In contrast, TMPRSS2 that is implicated in SARS-CoV-2 entry has been detected in all MSC samples. These results are of particular importance for future MSC-based cell therapies to treat severe cases after COVID-19 infection.

Both Specific Endothelial and Proximal Tubular Adam17 Deletion Protect against Diabetic Nephropathy.

ADAM17 is a disintegrin and metalloproteinase capable of cleaving the ectodomains of a diverse variety of molecules including TNF-α, TGF-α, L-selectin, and ACE2. We have previously demonstrated that renal ADAM17 is upregulated in diabetic mice. The role of endothelial (eAdam17) and proximal tubular (tAdam17) Adam17 deletion in renal histology, modulation of the renin angiotensin system (RAS), renal inflammation, and fibrosis was studied in a mouse model of type 1 Diabetes Mellitus. Moreover, the effect of Adam17 deletion in an in vitro 3D cell culture from human proximal tubular cells under high glucose conditions was evaluated. eAdam17 deletion attenuates renal fibrosis and inflammation, whereas tAdam17 deletion decreases podocyte loss, attenuates the RAS, and decreases macrophage infiltration, α-SMA and collagen accumulation. The 3D in vitro cell culture reinforced the findings obtained in tAdam17KO mice with decreased fibrosis in the Adam17 knockout spheroids. In conclusion, Adam17 deletion either in the endothelial or the tubular cells mitigates kidney injury in the diabetic mice by targeting different pathways. The manipulation of Adam17 should be considered as a therapeutic strategy for treating DN.

3D-microtissue derived secretome as a cell-free approach for enhanced mineralization of scaffolds in the chorioallantoic membrane model.

Bone regeneration is a complex process and the clinical translation of tissue engineered constructs (TECs) remains a challenge. The combination of biomaterials and mesenchymal stem cells (MSCs) may enhance the healing process through paracrine effects. Here, we investigated the influence of cell format in combination with a collagen scaffold on key factors in bone healing process, such as mineralization, cell infiltration, vascularization, and ECM production. MSCs as single cells (2D-SCs), assembled into microtissues (3D-MTs) or their corresponding secretomes were combined with a collagen scaffold and incubated on the chicken embryo chorioallantoic membrane (CAM) for 7 days. A comprehensive quantitative analysis was performed on a cellular level by histology and by microcomputed tomography (microCT). In all experimental groups, accumulation of collagen and glycosaminoglycan within the scaffold was observed over time. A pronounced cell infiltration and vascularization from the interface to the surface region of the CAM was detected. The 3D-MT secretome showed a significant mineralization of the biomaterial using microCT compared to all other conditions. Furthermore, it revealed a homogeneous distribution pattern of mineralization deposits in contrast to the cell-based scaffolds, where mineralization was only at the surface. Therefore, the secretome of MSCs assembled into 3D-MTs may represent an interesting therapeutic strategy for a next-generation bone healing concept.

Thermal conditioning improves quality and speed of keratinocyte sheet production for burn wound treatment.

BACKGROUND AIMS: Cultured patient-specific keratinocyte sheets have been used clinically since the 1970s for the treatment of large severe burns. However, despite significant developments in recent years, successful and sustainable treatment is still a challenge. Reliable, high-quality grafts with faster availability and a flexible time window for transplantation are required to improve clinical outcomes. METHODS: Keratinocytes are usually grown in vitro at 37°C. Given the large temperature differences in native skin tissue, the aim of the authors' study was to investigate thermal conditioning of keratinocyte sheet production. Therefore, the influence of 31°C, 33°C and 37°C on cell expansion and differentiation in terms of proliferation and sheet formation efficacy was investigated. In addition, the thermal effect on the biological status and thus the quality of the graft was assessed on the basis of the release of wound healing-related biofactors in various stages of graft development. RESULTS: The authors demonstrated that temperature is a decisive factor in the production of human keratinocyte sheets. By using specific temperature ranges, the authors have succeeded in optimizing the individual manufacturing steps. During the cell expansion phase, cultivation at 37°C was most effective. After 6 days of culture at 37°C, three times and six times higher numbers of viable cells were obtained compared with 33°C and 31°C. During the cell differentiation and sheet formation phase, however, the cells benefited from a mildly hypothermic temperature of 33°C. Keratinocytes showed increased differentiation potential and formed better epidermal structures, which led to faster biomechanical sheet stability at day 18. In addition, a cultivation temperature of 33°C resulted in a longer lasting and higher secretion of the investigated immunomodulatory, anti-inflammatory, angiogenic and pro-inflammatory biofactors. CONCLUSIONS: These results show that by using specific temperature ranges, it is possible to accelerate the large-scale production of cultivated keratinocyte sheets while at the same time improving quality. Cultivated keratinocyte sheets are available as early as 18 days post-biopsy and at any time for 7 days thereafter, which increases the flexibility of the process for surgeons and patients alike. These findings will help to provide better clinical outcomes, with an increased take rate in severe burn patients.

Geometry influences inflammatory host cell response and remodeling in tissue-engineered heart valves in-vivo.

Regenerative tissue-engineered matrix-based heart valves (TEM-based TEHVs) may become an alternative to currently-used bioprostheses for transcatheter valve replacement. We recently identified TEM-based TEHVs-geometry as one key-factor guiding their remodeling towards successful long-term performance or failure. While our first-generation TEHVs, with a simple, non-physiological valve-geometry, failed over time due to leaflet-wall fusion phenomena, our second-generation TEHVs, with a computational modeling-inspired design, showed native-like remodeling resulting in long-term performance. However, a thorough understanding on how TEHV-geometry impacts the underlying host cell response, which in return determines tissue remodeling, is not yet fully understood. To assess that, we here present a comparative samples evaluation derived from our first- and second-generation TEHVs. We performed an in-depth qualitative and quantitative (immuno-)histological analysis focusing on key-players of the inflammatory and remodeling cascades (M1/M2 macrophages, α-SMA+- and endothelial cells). First-generation TEHVs were prone to chronic inflammation, showing a high presence of macrophages and α-SMA+-cells, hinge-area thickening, and delayed endothelialization. Second-generation TEHVs presented with negligible amounts of macrophages and α-SMA+-cells, absence of hinge-area thickening, and early endothelialization. Our results suggest that TEHV-geometry can significantly influence the host cell response by determining the infiltration and presence of macrophages and α-SMA+-cells, which play a crucial role in orchestrating TEHV remodeling.

Identification and Validation of a Biomarker Signature in Patients With Resectable Pancreatic Cancer via Genome-Wide Screening for Functional Genetic Variants.

Importance: Surgery currently offers the only chance for a cure in pancreatic ductal adenocarcinoma (PDAC), but it carries a significant morbidity and mortality risk and results in varying oncologic outcomes. At present, to our knowledge, there are no tests available before surgical resection to identify tumors with an aggressive biological phenotype that could guide personalized treatment strategies. Objective: Identification of noninvasive genetic biomarkers that could direct therapy in patients whose cases are amenable to pancreatic cancer resection. Design, Setting, and Participants: This multicenter study combined a prospective European cohort of patients with PDAC who underwent pancreatic resection (from University Hospital of Zurich, Zurich, Switzerland; Cantonal Hospital of Winterthur, Winterthur, Switzerland; and University Clinic of Ulm, Ulm, Germany) with data from the Cancer Genome Atlas database in the United States, which includes prospectively registered patients with PDAC. A genome-wide screening for functional single-nucleotide polymorphisms (SNPs) that affect PDAC survival was conducted using the European cohort for identification and the Cancer Genome Atlas cohort for validation. We used Cox proportional hazards models to screen for high-frequency polymorphic variants that are associated with allelic differences in tumor-associated survival and either result in an altered protein structure and function or reside in known regulatory noncoding genomic regions. The false-discovery rate method was applied for multiple hypothesis-testing corrections. Data analysis occurred from November 2017 to May 2018. Exposures: Pancreatic resection. Main Outcomes and Measures: Tumor-associated survival. Results: A total of 195 patients in the European cohort were included, as well as 136 patients in the Cancer Genome Atlas cohort (overall median [range] age, 66 [19-87] years; 156 [47.1%] were women, and 175 [52.9%] were men). Two SNPs in noncoding, functional regions of genes that regulate cancer progression, invasion, and metastasis were identified (CHI3L2 SNP rs684559 and CD44 SNP rs353630). These were associated with survival after PDAC resection; patients who carry the risk alleles at 1 of both SNP loci had a 2.63-fold increased risk for tumor-associated death compared with those with protective genotypes (hazard ratio for survival, 0.38 [95% CI, 0.27-0.53]; P = 1.0 × 10-8). Conclusions and Relevance: The identified polymorphisms may serve as a noninvasive biomarker signature of prospective survival after pancreatic resection that is readily available at the time of PDAC diagnosis. This signature can be used to identify a subset of high-risk patients with PDAC with very low survival probability who might be eligible for inclusion in clinical trials of new therapeutic strategies, including neoadjuvant chemotherapy protocols. In addition, the biological knowledge about these SNPs could help guide the development of individualized genomic strategies for PDAC therapies.

Cellular self-assembly into 3D microtissues enhances the angiogenic activity and functional neovascularization capacity of human cardiopoietic stem cells.

While cell therapy has been proposed as next-generation therapy to treat the diseased heart, current strategies display only limited clinical efficacy. Besides the ongoing quest for the ideal cell type, in particular the very low retention rate of single-cell (SC) suspensions after delivery remains a major problem. To improve cellular retention, cellular self-assembly into 3D microtissues (MTs) prior to transplantation has emerged as an encouraging alternative. Importantly, 3D-MTs have also been reported to enhance the angiogenic activity and neovascularization potential of stem cells. Therefore, here using the chorioallantoic membrane (CAM) assay we comprehensively evaluate the impact of cell format (SCs versus 3D-MTs) on the angiogenic potential of human cardiopoietic stem cells, a promising second-generation cell type for cardiac repair. Biodegradable collagen scaffolds were seeded with human cardiopoietic stem cells, either as SCs or as 3D-MTs generated by using a modified hanging drop method. Thereafter, seeded scaffolds were placed on the CAM of living chicken embryos and analyzed for their perfusion capacity in vivo using magnetic resonance imaging assessment which was then linked to a longitudinal histomorphometric ex vivo analysis comprising blood vessel density and characteristics such as shape and size. Cellular self-assembly into 3D-MTs led to a significant increase of vessel density mainly driven by a higher number of neo-capillary formation. In contrast, SC-seeded scaffolds displayed a higher frequency of larger neo-vessels resulting in an overall 1.76-fold higher total vessel area (TVA). Importantly, despite that larger TVA in SC-seeded group, the mean perfusion capacity (MPC) was comparable between groups, therefore suggesting functional superiority together with an enhanced perfusion efficacy of the neo-vessels in 3D-MT-seeded scaffolds. This was further underlined by a 1.64-fold higher perfusion ratio when relating MPC to TVA. Our study shows that cellular self-assembly of human cardiopoietic stem cells into 3D-MTs substantially enhances their overall angiogenic potential and their functional neovascularization capacity. Hence, the concept of 3D-MTs may be considered to increase the therapeutic efficacy of future cell therapy concepts.

A Simple Modification Method to Obtain Anisotropic and Porous 3D Microfibrillar Scaffolds for Surgical and Biomedical Applications.

In native tissues, cellular organization is predominantly anisotropic. Yet, it remains a challenge to engineer anisotropic scaffolds that promote anisotropic cellular organization at macroscopic length scales. To overcome this challenge, an innovative, cheap and easy method to align clinically approved non-woven surgical microfibrillar scaffolds is presented. The method involves a three-step process of coating, unidirectional stretching of scaffolds after heating them above glass transition temperature, and cooling back to room temperature. Briefly, a polymer coating is applied to a non-woven mesh that results in a partial welding of randomly oriented microfibers at their intersection points. The coated scaffold is then heated above the glass transition temperature of the coating and the scaffold polymer. Subsequently, the coated scaffold is stretched to produce aligned and three dimentional (3D) porous fibrillar scaffolds. In a proof of concept study, a polyglycolic acid (PGA) micro-fibrillar scaffold was coated with poly(4-hydroxybutirate) (P4HB) acid and subsequently aligned. Fibroblasts were cultured in vitro within the scaffold and results showed an increase in cellular alignment along the direction of the PGA fibers. This method can be scaled up easily for industrial production of polymeric meshes or directly applied to small pieces of scaffolds at the point of care.

Induced pluripotent stem cells derived from human amnion in chemically defined conditions.

Fetal stem cells are a unique type of adult stem cells that have been suggested to be broadly multipotent with some features of pluripotency. Their clinical potential has been documented but their upgrade to full pluripotency could open up a wide range of cell-based therapies particularly suited for pediatric tissue engineering, longitudinal studies or disease modeling. Here we describe episomal reprogramming of mesenchymal stem cells from the human amnion to pluripotency (AM-iPSC) in chemically defined conditions. The AM-iPSC expressed markers of embryonic stem cells, readily formed teratomas with tissues of all three germ layers present and had a normal karyotype after around 40 passages in culture. We employed novel computational methods to determine the degree of pluripotency from microarray and RNA sequencing data in these novel lines alongside an iPSC and ESC control and found that all lines were deemed pluripotent, however, with variable scores. Differential expression analysis then identified several groups of genes that potentially regulate this variability in lines within the boundaries of pluripotency, including metallothionein proteins. By further studying this variability, characteristics relevant to cell-based therapies, like differentiation propensity, could be uncovered and predicted in the pluripotent stage.

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions.

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods - flow cytometry, confocal imaging, teratoma formation and transcriptional profiling - is also described. The newly generated lines express markers of embryonic stem cells - Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 - while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

Monitoring and manipulating cellular crosstalk during kidney fibrosis inside a 3D in vitro co-culture.

In pharmacological research the development of promising lead compounds requires a detailed understanding of the dynamics of disease progression. However, for many diseases, such as kidney fibrosis, gaining such understanding requires complex real-time, multi-dimensional analysis of diseased and healthy tissue. To allow for such studies with increased throughput we established a dextran hydrogel-based in vitro 3D co-culture as a disease model for kidney fibrosis aimed at the discovery of compounds modulating the epithelial/mesenchymal crosstalk. This platform mimics a simplified pathological renal microenvironment at the interface between tubular epithelial cells and surrounding quiescent fibroblasts. We combined this 3D technology with epithelial reporter cell lines expressing fluorescent biomarkers in order to visualize pathophysiological cell state changes resulting from toxin-mediated chemical injury. Epithelial cell damage onset was robustly detected by image-based monitoring, and injured epithelial spheroids induced myofibroblast differentiation of co-cultured quiescent human fibroblasts. The presented 3D co-culture system therefore provides a unique model system for screening of novel therapeutic molecules capable to interfere and modulate the dialogue between epithelial and mesenchymal cells.

Clot-entrapped blood cells in synergy with human mesenchymal stem cells create a pro-angiogenic healing response.

Blood clots stop bleeding and provide cell-instructive microenvironments. Still, in vitro models used to study implant performance typically neglect any possible interactions of recruited cells with surface-adhering blood clots. Here we study the interaction and synergies of bone marrow derived human mesenchymal stem cells (hMSCs) with surface-induced blood clots in an in vitro model by fluorescence microscopy, scanning and correlative light and electron microscopy, ELISA assays and zymography. The clinically used alkali-treated rough titanium (Ti) surfaces investigated here are known to enhance blood clotting compared to native Ti and to improve the healing response, but the underlying mechanisms remain elusive. Here we show that the presence of blood clots synergistically increased hMSC proliferation, extracellular matrix (ECM) remodelling and the release of matrix fragments and angiogenic VEGF, but did not increase the osteogenic differentiation of hMSCs. While many biomaterials are nowadays engineered to release pro-angiogenic factors, we show here that clot-entrapped blood cells on conventional materials in synergy with hMSCs are potent producers of pro-angiogenic factors. Our data might thus not only explain why alkali-treatment is beneficial for Ti implant integration, but they suggest that the physiological importance of blood clots to create pro-angiogenic environments on implants has been greatly underestimated. The importance of blood clots might have been missed because the pro-angiogenic functions get activated only upon stimulation by synergistic interactions with the invading cells.

High heparin content surface-modified polyurethane discs promote rapid and stable angiogenesis in full thickness skin defects through VEGF immobilization.

Three-dimensional scaffolds have the capacity to serve as an architectural framework to guide and promote tissue regeneration. Parameters such as the type of material, growth factors, and pore dimensions are therefore critical in the scaffold's success. In this study, heparin has been covalently bound to the surface of macroporous polyurethane (PU) discs via two different loading methods to determine if the amount of heparin content had an influence on the therapeutic affinity loading and release of (VEGF165 ) in full thickness skin defects. PU discs (5.4 mm diameter, 300 µm thickness, and interconnected pore size of 150 µm) were produced with either a low (2.5 mg/g) or high (6.6 mg/g) heparin content (LC and HC respectively), and were implanted into the modified dorsal skin chamber (MDSC) of C57BL/6 J mice with and without VEGF. Both low- and high-content discs with immobilized VEGF165 (LCV and HCV, respectively) presented accelerated neovascularization and tissue repair in comparison to heparin discs alone. However, the highest angiogenetic peak was on day 7 with subsequent stabilization for HCV, whereas other groups displayed a delayed peak on day 14. We therefore attribute the superior performance of HCV due to its ability to hold more VEGF165, based on its increased heparin surface coverage, as also demonstrated in VEGF elution dynamics. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2543-2550, 2017.

Sequential activation of different pathway networks in ischemia-affected and non-affected myocardium, inducing intrinsic remote conditioning to prevent left ventricular remodeling.

We have analyzed the pathway networks of ischemia-affected and remote myocardial areas after repetitive ischemia/reperfusion (r-I/R) injury without ensuing myocardial infarction (MI) to elaborate a spatial- and chronologic model of cardioprotective gene networks to prevent left ventricular (LV) adverse remodeling. Domestic pigs underwent three cycles of 10/10 min r-I/R by percutaneous intracoronary balloon inflation/deflation in the mid left anterior descending artery, without consecutive MI. Sham interventions (n = 8) served as controls. Hearts were explanted at 5 h (n = 6) and 24 h (n = 6), and transcriptomic profiling of the distal (ischemia-affected) and proximal (non-affected) anterior myocardial regions were analyzed by next generation sequencing (NGS) and post-processing with signaling pathway impact and pathway network analyses. In ischemic region, r-I/R induced early activation of Ca-, adipocytokine and insulin signaling pathways with key regulator STAT3, which was also upregulated in the remote areas together with clusterin (CLU) and TNF-alpha. During the late phase of cardioprotection, antigen immunomodulatory pathways were activated with upregulation of STAT1 and CASP3 and downregulation of neprilysin in both zones, suggesting r-I/R induced intrinsic remote conditioning. The temporo-spatially differently activated pathways revealed a global myocardial response, and neprilysin and the STAT family as key regulators of intrinsic remote conditioning for prevention of adverse remodeling.

Safety and efficacy of cardiopoietic stem cells in the treatment of post-infarction left-ventricular dysfunction - From cardioprotection to functional repair in a translational pig infarction model.

To date, clinical success of cardiac cell-therapies remains limited. To enhance the cardioreparative properties of stem cells, the concept of lineage-specification through cardiopoietic-guidance has been recently suggested. However, so far, only results from murine studies and from a clinical pilot-trial in chronic heart-failure (CHF) are available, while systematic evidence of its therapeutic-efficacy is still lacking. Importantly, also no data from large animals or for other indications are available. Therefore, we here investigate the therapeutic-efficacy of human cardiopoietic stem cells in the treatment of post-infarction LV-dysfunction using a translational pig-model. Using growth-factor priming, lineage-specification of human bone-marrow derived MSCs was achieved to generate cardiopoietic stem cells according to GMP-compliant protocols. Thereafter, pigs with post-infarction LV-dysfunction (sub-acute phase;1-month) were randomized to either receive transcatheter NOGA 3D electromechanical-mapping guided intramyocardial transplantation of cardiopoietic cells or saline (control). After 30days, cardiac MRI (cMRI) was performed for functional evaluation and in-vivo cell-tracking. This approach was coupled with a comprehensive post-mortem cell-fate and mode-of-repair analysis. Cardiopoietic cell therapy was safe and ejection-fraction was significantly higher when compared to controls (p = 0.012). It further prevented maladaptive LV-remodeling and revealed a significantly lower relative and total infarct-size (p = 0.043 and p = 0.012). As in-vivo tracking and post-mortem analysis displayed only limited intramyocardial cardiopoietic cell-integration, the significant induction of neo-angiogenesis (∼40% higher; p = 0.003) and recruitment of endogenous progenitors (∼2.5x higher; p = 0.008) to the infarct border-zone appeared to be the major modes-of-repair. This is the first report using a pre-clinical large animal-model to demonstrate the safety and efficacy of cardiopoietic stem cells for the treatment of post-infarction LV-dysfunction to prevent negative LV-remodeling and subsequent CHF. It further provides insight into post-delivery cardiopoietic cell-fate and suggests the mechanisms of cardiopoietic cell-induced cardiac-repair. The adoption of GMP-/GLP-compliant methodologies may accelerate the translation into a phase-I clinical-trial in patients with post-ischemic LV-dysfunction broadening the current indication of this interesting cell-type.

Nogo-A regulates vascular network architecture in the postnatal brain.

Recently, we discovered a new role for the well-known axonal growth inhibitory molecule Nogo-A as a negative regulator of angiogenesis in the developing central nervous system. However, how Nogo-A affected the three-dimensional (3D) central nervous system (CNS) vascular network architecture remained unknown. Here, using vascular corrosion casting, hierarchical, synchrotron radiation µCT-based network imaging and computer-aided network analysis, we found that genetic ablation of Nogo-A significantly increased the three-dimensional vascular volume fraction in the postnatal day 10 (P10) mouse brain. More detailed analysis of the cerebral cortex revealed that this effect was mainly due to an increased number of capillaries and capillary branchpoints. Interestingly, other vascular parameters such as vessel diameter, -length, -tortuosity, and -volume were comparable between both genotypes for non-capillary vessels and capillaries. Taken together, our three-dimensional data showing more vessel segments and branchpoints at unchanged vessel morphology suggest that stimulated angiogenesis upon Nogo-A gene deletion results in the insertion of complete capillary micro-networks and not just single vessels into existing vascular networks. These findings significantly enhance our understanding of how angiogenesis, vascular remodeling, and three-dimensional vessel network architecture are regulated during central nervous system development. Nogo-A may therefore be a potential novel target for angiogenesis-dependent central nervous system pathologies such as brain tumors or stroke.