No Clinical Efficacy of Adipose-Derived Regenerative Cells and Lipotransfer in Breast Cancer-Related Lymphedema: A double-blinded placebo-controlled phase-II trial.

BACKGROUND: Breast cancer-related lymphedema (BCRL) is a debilitating sequela affecting up to one in three breast cancer survivors. Current treatments are palliative and does not address the underlying lymphatic injury. Recently, preclinical and non-randomized studies have shown promising results using Adipose-Derived Regenerative Cells (ADRCs) and lipotransfer in alleviating BCRL through regeneration of lymphatic tissue. However no randomized controlled trial has been performed in an attempt to eliminate a placebo effect. METHODS: This randomized, double-blinded, placebo-controlled trial included patients with no-option, persistent disabling unilateral BCRL. Patients were randomly assigned in a 1:1 ratio to receive either autologous ADRCs (4.20x10 7±1.75x10 7 cells) and 30cc lipotransfer or placebo (saline) to the axilla. The primary outcome was a change in BCRL volume one year after treatment. Secondary outcomes included changes in the quality of life, indocyanine green lymphangiography stage, bioimpedance, and safety. RESULTS: Eighty patients were included, of which 39 were allocated to ADRCs and lipotransfer treatment and 41 to placebo treatment. Baseline characteristics were similar in both groups. One year after treatment, no objective improvements were observed in the treatment or placebo groups. In contrast, significant subjective improvements were noted for both the treatment and placebo groups. CONCLUSION: This trial failed to confirm a benefit of ADRCs and lipotransfer in the treatment of BCRL. These non-confirmatory results suggest that ADRC and lipotransfer should not be recommended for alleviating BCRL at this time. However, we cannot exclude that repeated treatments or higher doses of ADRCs or lipotransfer could yield a clinical effect.

Transfer of cardiomyocyte-derived extracellular vesicles to neighboring cardiac cells requires tunneling nanotubes during heart development.

Rationale: Extracellular vesicles (EVs) are thought to mediate intercellular communication during development and disease. Yet, biological insight to intercellular EV transfer remains elusive, also in the heart, and is technically challenging to demonstrate. Here, we aimed to investigate biological transfer of cardiomyocyte-derived EVs in the neonatal heart. Methods: We exploited CD9 as a marker of EVs, and generated two lines of cardiomyocyte specific EV reporter mice: Tnnt2-Cre; double-floxed inverted CD9/EGFP and αMHC-MerCreMer; double-floxed inverted CD9/EGFP. The two mouse lines were utilized to determine whether developing cardiomyocytes transfer EVs to other cardiac cells (non-myocytes and cardiomyocytes) in vitro and in vivo and investigate the intercellular transport pathway of cardiomyocyte-derived EVs. Results: Genetic tagging of cardiomyocytes was confirmed in both reporter mouse lines and proof of concept in the postnatal heart showed that, a fraction of EGFP+/MYH1- non-myocytes exist firmly demonstrating in vivo cardiomyocyte-derived EV transfer. However, two sets of direct and indirect EGFP +/- cardiac cell co-cultures showed that cardiomyocyte-derived EGFP+ EV transfer requires cell-cell contact and that uptake of EGFP+ EVs from the medium is limited. The same was observed when co-cultiring with mouse macrophages. Further mechanistic insight showed that cardiomyocyte EV transfer occurs through type I tunneling nanotubes. Conclusion: While the current notion assumes that EVs are transferred through secretion to the surroundings, our data show that cardiomyocyte-derived EV transfer in the developing heart occurs through nanotubes between neighboring cells. Whether these data are fundamental and relate to adult hearts and other organs remains to be determined, but they imply that the normal developmental process of EV transfer goes through cell-cell contact rather than through the extracellular compartment.

Ploidy-stratified single cardiomyocyte transcriptomics map Zinc Finger E-Box Binding Homeobox 1 to underly cardiomyocyte proliferation before birth.

Whereas cardiomyocytes (CMs) in the fetal heart divide, postnatal CMs fail to undergo karyokinesis and/or cytokinesis and therefore become polyploid or binucleated, a key process in terminal CM differentiation. This switch from a diploid proliferative CM to a terminally differentiated polyploid CM remains an enigma and seems an obstacle for heart regeneration. Here, we set out to identify the transcriptional landscape of CMs around birth using single cell RNA sequencing (scRNA-seq) to predict transcription factors (TFs) involved in CM proliferation and terminal differentiation. To this end, we established an approach combining fluorescence activated cell sorting (FACS) with scRNA-seq of fixed CMs from developing (E16.5, P1, and P5) mouse hearts, and generated high-resolution single-cell transcriptomic maps of in vivo diploid and tetraploid CMs, increasing the CM resolution. We identified TF-networks regulating the G2/M phases of developing CMs around birth. ZEB1 (Zinc Finger E-Box Binding Homeobox 1), a hereto unknown TF in CM cell cycling, was found to regulate the highest number of cell cycle genes in cycling CMs at E16.5 but was downregulated around birth. CM ZEB1-knockdown reduced proliferation of E16.5 CMs, while ZEB1 overexpression at P0 after birth resulted in CM endoreplication. These data thus provide a ploidy stratified transcriptomic map of developing CMs and bring new insight to CM proliferation and endoreplication identifying ZEB1 as a key player in these processes.

The Inflammatory Response after Moderate Contusion Spinal Cord Injury: A Time Study.

Spinal cord injury (SCI) initiates detrimental cellular and molecular events that lead to acute and delayed neuroinflammation. Understanding the role of the inflammatory response in SCI requires insight into the temporal and cellular synthesis of inflammatory mediators. We subjected C57BL/6J mice to SCI and investigated inflammatory reactions. We examined activation, recruitment, and polarization of microglia and infiltrating immune cells, focusing specifically on tumor necrosis factor (TNF) and its receptors TNFR1 and TNFR2. In the acute phase, TNF expression increased in glial cells and neuron-like cells, followed by infiltrating immune cells. TNFR1 and TNFR2 levels increased in the delayed phase and were found preferentially on neurons and glial cells, respectively. The acute phase was dominated by the infiltration of granulocytes and macrophages. Microglial/macrophage expression of Arg1 increased from 1-7 days after SCI, followed by an increase in Itgam, Cx3cr1, and P2ry12, which remained elevated throughout the study. By 21 and 28 days after SCI, the lesion core was populated by galectin-3+, CD68+, and CD11b+ microglia/macrophages, surrounded by a glial scar consisting of GFAP+ astrocytes. Findings were verified in postmortem tissue from individuals with SCI. Our findings support the consensus that future neuroprotective immunotherapies should aim to selectively neutralize detrimental immune signaling while sustaining pro-regenerative processes.

Adipose-Derived Stem Cells from Type 2 Diabetic Rats Retain Positive Effects in a Rat Model of Erectile Dysfunction.

Erectile dysfunction is a common complication associated with type 2 diabetes mellitus (T2DM) and after prostatectomy in relation to cancer. The regenerative effect of cultured adipose-derived stem cells (ASCs) for ED therapy has been documented in multiple preclinical trials as well as in recent Pase 1 trials in humans. However, some studies indicate that diabetes negatively affects the mesenchymal stem cell pool, implying that ASCs from T2DM patients could have impaired regenerative capacity. Here, we directly compared ASCs from age-matched diabetic Goto-Kakizaki (ASCGK) and non-diabetic wild type rats (ASCWT) with regard to their phenotypes, proteomes and ability to rescue ED in normal rats. Despite ASCGK exhibiting a slightly lower proliferation rate, ASCGK and ASCWT proteomes were more or less identical, and after injections to corpus cavernosum they were equally efficient in restoring erectile function in a rat ED model entailing bilateral nerve crush injury. Moreover, molecular analysis of the corpus cavernosum tissue revealed that both ASCGK and ASCWT treated rats had increased induction of genes involved in recovering endothelial function. Thus, our finding argues that T2DM does not appear to be a limiting factor for autologous adipose stem cell therapy when correcting for ED.

Early-stage inflammation changes in supraspinatus muscle after rotator cuff tear.

BACKGROUND: Rotator cuff (RC) tendon tear leads to impaired shoulder function and pain. The supraspinatus (SS) tendon is most often affected, but the biological response of the SS muscle to SS tendon tear is largely unknown. This study aimed to investigate time-dependent muscle inflammation, degeneration, fatty infiltration, and regeneration in experimental SS tear conditions. METHODS: Forty-five C57BL/6 mice were subjected to SS tendon tear and allowed to recover for 1, 3, 5, 7, 14, or 28 days. The extent of muscle damage was examined using histologic, flow cytometric, proteomic, and chemiluminescence analyses. RESULTS: We found that muscle inflammation peaked around day 5 with increased monocyte infiltration and increased cytokine levels in the ipsilateral compared to the contralateral SS muscle. Bioinformatics analysis of proteomics on mice that survived 5 days after RC tendon tear revealed upregulated proteins involved in "neutrophil activation involved in immune response" and "extracellular matrix organization," whereas "skeletal muscle tissue development and contraction" and "respiratory electron transport chain" were among the most downregulated. Histologic analysis of collagen showed increased collagen accumulation and fatty infiltration of the ipsilateral SS over time. Finally, we observed time- and lesion-dependent changes in satellite cell and fibro-adipogenic progenitor populations. CONCLUSION: Altogether, we demonstrate that the SS muscle shows severe signs of acute inflammation, early degeneration, and fatty infiltration, as well as reduced regenerative potential following SS tendon tear.

Decellularised Human Umbilical Artery as a Vascular Graft Elicits Minimal Pro-Inflammatory Host Response Ex Vivo and In Vivo.

Small diameter (<6 mm) vessel grafts still pose a challenge for scientists worldwide. Decellularised umbilical artery (dUA) remains promising as small diameter tissue engineered vascular graft (TEVG), yet their immunogenicity remains unknown. Herein, we evaluated the host immune responses, with a focus on the innate part, towards human dUA implantation in mice, and confirmed our findings in an ex vivo allogeneic human setup. Overall, we did not observe any differences in the number of circulating white blood cells nor the number of monocytes among three groups of mice (1) dUA patch; (2) Sham; and (3) Mock throughout the study (day -7 to 28). Likewise, we found no difference in systemic inflammatory and anti-inflammatory cytokine levels between groups. However, a massive local remodelling response with M2 macrophages were observed in the dUA at day 28, whereas M1 macrophages were less frequent. Moreover, human monocytes from allogeneic individuals were differentiated into macrophages and exposed to lyophilised dUA to maximize an eventual M1 response. Yet, dUA did not elicit any immediate M1 response as determined by the absence of CCR7 and CXCL10. Together this suggests that human dUA elicits a minimal pro-inflammatory response further supporting its use as a TEVG in an allogeneic setup.

Apex Resection in Zebrafish (Danio rerio) as a Model of Heart Regeneration: A Video-Assisted Guide.

Ischemic heart disease is one of the leading causes of deaths worldwide. A major hindrance to resolving this challenge lies in the mammalian hearts inability to regenerate after injury. In contrast, zebrafish retain a regenerative capacity of the heart throughout their lifetimes. Apex resection (AR) is a popular zebrafish model for studying heart regeneration, and entails resecting 10-20% of the heart in the apex region, whereafter the regeneration process is monitored until the heart is fully regenerated within 60 days. Despite this popularity, video tutorials describing this technique in detail are lacking. In this paper we visualize and describe the entire AR procedure including anaesthesia, surgery, and recovery. In addition, we show that the concentration and duration of anaesthesia are important parameters to consider, to balance sufficient levels of sedation and minimizing mortality. Moreover, we provide examples of how zebrafish heart regeneration can be assessed both in 2D (immunohistochemistry of heart sections) and 3D (analyses of whole, tissue cleared hearts using multiphoton imaging). In summary, this paper aims to aid beginners in establishing and conducting the AR model in their laboratory, but also to spur further interest in improving the model and its evaluation.

Review: Tissue Engineering of Small-Diameter Vascular Grafts and Their In Vivo Evaluation in Large Animals and Humans.

To date, a wide range of materials, from synthetic to natural or a mixture of these, has been explored, modified, and examined as small-diameter tissue-engineered vascular grafts (SD-TEVGs) for tissue regeneration either in vitro or in vivo. However, very limited success has been achieved due to mechanical failure, thrombogenicity or intimal hyperplasia, and improvements of the SD-TEVG design are thus required. Here, in vivo studies investigating novel and relative long (10 times of the inner diameter) SD-TEVGs in large animal models and humans are identified and discussed, with emphasis on graft outcome based on model- and graft-related conditions. Only a few types of synthetic polymer-based SD-TEVGs have been evaluated in large-animal models and reflect limited success. However, some polymers, such as polycaprolactone (PCL), show favorable biocompatibility and potential to be further modified and improved in the form of hybrid grafts. Natural polymer- and cell-secreted extracellular matrix (ECM)-based SD-TEVGs tested in large animals still fail due to a weak strength or thrombogenicity. Similarly, native ECM-based SD-TEVGs and in-vitro-developed hybrid SD-TEVGs that contain xenogeneic molecules or matrix seem related to a harmful graft outcome. In contrast, allogeneic native ECM-based SD-TEVGs, in-vitro-developed hybrid SD-TEVGs with allogeneic banked human cells or isolated autologous stem cells, and in-body tissue architecture (IBTA)-based SD-TEVGs seem to be promising for the future, since they are suitable in dimension, mechanical strength, biocompatibility, and availability.

Adipose-derived regenerative cells and lipotransfer in alleviating breast cancer-related lymphedema: An open-label phase I trial with 4 years of follow-up.

Patients with breast cancer-related lymphedema (BCRL) have reduced quality of life and arm function. Current treatments are palliative, and treatments improving lymphedema are lacking. Preclinical studies have suggested that adipose-derived regenerative cells (ADRCs) can alleviate lymphedema. We, therefore, aimed to assess whether ADRCs can alleviate lymphedema in clinical reality with long-term follow-up. We treated 10 patients with BCRL using ADRCs and a scar-releasing lipotransfer to the axillary region, and all patients were followed 1, 3, 6, 12, and 48 months after treatment. The primary endpoint was change in arm volume. Secondary endpoints were safety, change in lymphedema symptoms, quality of life, lymphedema-associated cellulitis, and conservative treatment use. There was no significant decrease in BCRL volume after treatment. However, self-reported upper extremity disability and arm heaviness and tension improved. Six patients reduced their use of conservative BCRL treatment. Five patients felt that their BCRL had improved substantially, and four of these would redo the treatment. We did not observe any cases of locoregional breast cancer recurrence. In this phase I study with 4 years of follow-up, axillary delivered ADRCs and lipotransfer were safe and feasible and improved BCRL symptoms and upper extremity function. Randomized controlled trials are needed to confirm the results of this study.

Conditional Ablation of Myeloid TNF Improves Functional Outcome and Decreases Lesion Size after Spinal Cord Injury in Mice.

Spinal cord injury (SCI) is a devastating condition consisting of an instant primary mechanical injury followed by a secondary injury that progresses for weeks to months. The cytokine tumor necrosis factor (TNF) plays an important role in the pathophysiology of SCI. We investigated the effect of myeloid TNF ablation (peripheral myeloid cells (macrophages and neutrophils) and microglia) versus central myeloid TNF ablation (microglia) in a SCI contusion model. We show that TNF ablation in macrophages and neutrophils leads to reduced lesion volume and improved functional outcome after SCI. In contrast, TNF ablation in microglia only or TNF deficiency in all cells had no effect. TNF levels tended to be decreased 3 h post-SCI in mice with peripheral myeloid TNF ablation and was significantly decreased 3 days after SCI. Leukocyte and microglia populations and all other cytokines (IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, and IFNγ) and chemokines (CCL2, CCL5, and CXCL1) investigated, in addition to TNFR1 and TNFR2, were comparable between genotypes. Analysis of post-SCI signaling cascades demonstrated that the MAPK kinase SAPK/JNK decreased and neuronal Bcl-XL levels increased post-SCI in mice with ablation of TNF in peripheral myeloid cells. These findings demonstrate that peripheral myeloid cell-derived TNF is pathogenic in SCI.

A Systematic Exposition of Methods used for Quantification of Heart Regeneration after Apex Resection in Zebrafish.

Myocardial infarction (MI) is a worldwide condition that affects millions of people. This is mainly caused by the adult human heart lacking the ability to regenerate upon injury, whereas zebrafish have the capacity through cardiomyocyte proliferation to fully regenerate the heart following injury such as apex resection (AR). But a systematic overview of the methods used to evidence heart regrowth and regeneration in the zebrafish is lacking. Herein, we conducted a systematical search in Embase and Pubmed for studies on heart regeneration in the zebrafish following injury and identified 47 AR studies meeting the inclusion criteria. Overall, three different methods were used to assess heart regeneration in zebrafish AR hearts. 45 out of 47 studies performed qualitative (37) and quantitative (8) histology, whereas immunohistochemistry for various cell cycle markers combined with cardiomyocyte specific proteins was used in 34 out of 47 studies to determine cardiomyocyte proliferation qualitatively (6 studies) or quantitatively (28 studies). For both methods, analysis was based on selected heart sections and not the whole heart, which may bias interpretations. Likewise, interstudy comparison of reported cardiomyocyte proliferation indexes seems complicated by distinct study designs and reporting manners. Finally, six studies performed functional analysis to determine heart function, a hallmark of human heart injury after MI. In conclusion, our data implies that future studies should consider more quantitative methods eventually taking the 3D of the zebrafish heart into consideration when evidencing myocardial regrowth after AR. Furthermore, standardized guidelines for reporting cardiomyocyte proliferation and sham surgery details may be considered to enable inter study comparisons and robustly determine the effect of given genes on the process of heart regeneration.

Concise Review: Patency of Small-Diameter Tissue-Engineered Vascular Grafts: A Meta-Analysis of Preclinical Trials.

Several patient groups undergoing small-diameter (<6 mm) vessel bypass surgery have limited autologous vessels for use as grafts. Tissue-engineered vascular grafts (TEVG) have been suggested as an alternative, but the ideal TEVG remains to be generated, and a systematic overview and meta-analysis of clinically relevant studies is lacking. We systematically searched PubMed and Embase databases for (pre)clinical trials and identified three clinical and 68 preclinical trials ([>rabbit]; 873 TEVGs) meeting the inclusion criteria. Preclinical trials represented low to medium risk of bias, and binary logistic regression revealed that patency was significantly affected by recellularization, TEVG length, TEVG diameter, surface modification, and preconditioning. In contrast, scaffold types were less important. The patency was 63.5%, 89%, and 100% for TEVGs with a median diameter of 3 mm, 4 mm, and 5 mm, respectively. In the group of recellularized TEVGs, patency was not improved by using smooth muscle cells in addition to endothelial cells nor affected by the endothelial origin, but seems to benefit from a long-term (46-240 hours) recellularization time. Finally, data showed that median TEVG length (5 cm) and median follow-up (56 days) used in preclinical settings are relatively inadequate for direct clinical translation. In conclusion, our data imply that future studies should consider a TEVG design that at least includes endothelial recellularization and bioreactor preconditioning, and we suggest that more standard guidelines for testing and reporting TEVGs in large animals should be considered to enable interstudy comparisons and favor a robust and reproducible outcome as well as clinical translation.

A 12-Month Follow-up After a Single Intracavernous Injection of Autologous Adipose-Derived Regenerative Cells in Patients with Erectile Dysfunction Following Radical Prostatectomy: An Open-Label Phase I Clinical Trial.

OBJECTIVE: To explore safety in adipose-derived regenerative cells (ADRC) therapy, treating erectile dysfunction (ED). METHODS: Twenty-one patients with ED after radical prostatectomy, with no signs of recovery using conventional therapy, received a single intracavernous injection of autologous ADRC and were followed for 1 year. Six men were incontinent, and 15 were continent at inclusion. The primary (safety of ADRC therapy) and secondary endpoints (sexual function) were evaluated at 1, 3, 6, and 12 months after ADRC injection by registration of adverse events and validated questionnaires using the international index of erectile function-5 and erection hardness score. RESULTS: No serious adverse events occurred, but 8 reversible minor events related to the liposuction were noted. Eight out of 15 (53%) patients in the continent group reported erectile function sufficient for intercourse at 12 months. Baseline median international index of erectile function-5 scores (6.0; interquartile range [IQR] 3) were unchanged 1 month after the treatment, but significantly increased after 6 to 7 (IQR 17). This effect was sustained at 12 months (median 8; IQR 14). We did not see any improvements in erectile function in the group of incontinent men or among men with ED prior to radical prostatectomy. CONCLUSION: Intracavernous injection of ADRC is safe in this phase 1 study with a 12-month follow-up.

Human and Autologous Adipose-derived Stromal Cells Increase Flap Survival in Rats Independently of Host Immune Response.

INTRODUCTION: There is a rising interest in adipose-derived stromal cells for clinical use; however, it is unknown whether freshly isolated stromal cells (SVF) or culture-expanded cells (ASCs) are more efficacious. We therefore aimed to compare the 2 cellular therapies in an in vivo model of angiogenesis, the ischemic flap in rats, which induces acute ischemia. We also aimed to determine the importance of cell presence and the host immune response. METHODS: A total of 96 rats (n = 12 in each group) were used, and in each rat, a caudally based random flap measuring 2 × 7 cm was made. The study was conducted in 3 phases. First, each rat was treated with human SVF cells, human ASCs, or vehicle. Second, each rat was treated with human SVF, human SVF lysate, or vehicle. Finally, each rat was treated with rat (autologous) SVF cells or vehicle. Flap survival, vessel density, and stromal cell retention were evaluated after 7 days. RESULTS: The mean survival rates for SVF treatment regardless of human or autologous origin were significantly increased as compared with the control group. Adipose stem/stromal cell and SVF lysate injection did not increase flap survival. Vessel density was increased for human and rat SVF and human ASC but not for SVF lysate. Human cells were not detected in the flaps after 7 days. CONCLUSIONS: Flap survival increased with SVF treatment regardless of human or autologous origin, suggesting that increased flap survival is independent of the host immune response. All cell injections lead to increased vessel density, but it did not necessarily lead to increased flap survival. Further research should elaborate which molecular events make SVF treatment more efficacious than ASC.

Treatment of Breast Cancer-Related Lymphedema with Adipose-Derived Regenerative Cells and Fat Grafts: A Feasibility and Safety Study.

Breast cancer-related lymphedema (BCRL) is a debilitating late complication with a lack of treatment opportunities. Recent studies have suggested that mesenchymal stromal cells can alleviate lymphedema. Herein, we report the results from the first human pilot study with freshly isolated adipose-derived regenerative cells (ADRC) for treating lymphedema with 6 months follow-up. Ten BCRL patients were included. ADRC was injected directly into the axillary region, which was combined with a scar-releasing fat graft procedure. Primary endpoints were change in arm volume. Secondary endpoints were change in patient reported outcome and safety. The study is registered with ClinicalTrials.gov (NCT02592213). During follow-up, a small volume reduction was noted but was not significant. Five patients reduced their use of conservative management. Patient-reported outcomes improved significantly over time. ADRCs were well tolerated and only minor transient adverse events related to liposuction were noted. In this pilot study, a single injection of ADRC improved lymphedema based on patient-reported outcome measures, and there were no serious adverse events in the 6 months follow-up period. In addition, half of the patients reduced their use of conservative management. ADRC therapy is a promising interventional therapy for alleviating lymphedema, but results need to be confirmed in randomized clinical trials. Stem Cells Translational Medicine 2017;6:1666-1672.

The non-canonical NOTCH1 ligand Delta-like 1 homolog (DLK1) self interacts in mammals.

Delta-like 1 homolog (DLK1) is an imprinted gene, which is widely expressed during mammalian development and plays a pivotal role in differentiation of various tissue types. Most recently, we have shown that DLK1 interacts with NOTCH1, yet several Notch independent mechanisms have previously been suggested as well, but only poorly confirmed in a mammalian context. In the present study, we employed the mammalian two-hybrid (MTH) system, a genetic in vivo protein-protein interaction system, to show robust DLK1-DLK1, DLK1-FnI (Fibronectin) and DLK1-CFR (cysteine-rich FGF receptor) interactions, whereas the proposed DLK1-IGFBP1 interaction was not supported by MTH. Very little has previously been described on the DLK1 self-interaction. Herein, we showed by immunoprecipitation as well as Sulfo-SBED label transfer that the DLK1-DLK1 interaction likely is part of Dlk1's function in preadipocytes. Furthermore our data suggest that DLK1 interacts with itself through EGF domain 4 and 5, which is distinct from the recently described NOTCH1-DLK1 interaction, which occurs between EGF domain 5 and 6. This opens up the possibility that Notch independent mechanisms like the DLK1-DLK1 interaction may modulate the non-canonical NOTCH1-DLK1 interaction further complexing this system.

Safety and Potential Effect of a Single Intracavernous Injection of Autologous Adipose-Derived Regenerative Cells in Patients with Erectile Dysfunction Following Radical Prostatectomy: An Open-Label Phase I Clinical Trial.

BACKGROUND: Prostate cancer is the most common cancer in men, and radical prostatectomy (RP) often results in erectile dysfunction (ED) and a substantially reduced quality of life. The efficacy of current interventions, principal treatment with PDE-5 inhibitors, is not satisfactory and this condition presents an unmet medical need. Preclinical studies using adipose-derived stem cells to treat ED have shown promising results. Herein, we report the results of a human phase 1 trial with autologous adipose-derived regenerative cells (ADRCs) freshly isolated after a liposuction. METHODS: Seventeen men suffering from post RP ED, with no recovery using conventional therapy, were enrolled in a prospective phase 1 open-label and single-arm study. All subjects had RP performed 5-18 months before enrolment, and were followed for 6 months after intracavernosal transplantation. ADRCs were analyzed for the presence of stem cell surface markers, viability and ability to differentiate. Primary endpoint was the safety and tolerance of the cell therapy while the secondary outcome was improvement of erectile function. Any adverse events were reported and erectile function was assessed by IIEF-5 scores. The study is registered with ClinicalTrials.gov, NCT02240823. FINDINGS: Intracavernous injection of ADRCs was well-tolerated and only minor events related to the liposuction and cell injections were reported at the one-month evaluation, but none at later time points. Overall during the study period, 8 of 17 men recovered their erectile function and were able to accomplish sexual intercourse. Post-hoc stratification according to urinary continence status was performed. Accordingly, for continent men (median IIEFinclusion = 7 (95% CI 5-12), 8 out of 11 men recovered erectile function (IIEF6months = 17 (6-23)), corresponding to a mean difference of 0.57 (0.38-0.85; p = 0.0069), versus inclusion. In contrast, incontinent men did not regain erectile function (median IIEF1/3/6 months = 5 (95% CI 5-6); mean difference 1 (95% CI 0.85-1.18), p > 0.9999). INTERPRETATION: In this phase I trial a single intracavernosal injection of freshly isolated autologous ADRCs was a safe procedure. A potential efficacy is suggested by a significant improvement in IIEF-5 scores and erectile function. We suggest that ADRCs represent a promising interventional therapy of ED following prostatectomy. FUNDING: Danish Medical Research Council, Odense University Hospital and the Danish Cancer Society.

Do neonatal mouse hearts regenerate following heart apex resection?

The mammalian heart has generally been considered nonregenerative, but recent progress suggests that neonatal mouse hearts have a genuine capacity to regenerate following apex resection (AR). However, in this study, we performed AR or sham surgery on 400 neonatal mice from inbred and outbred strains and found no evidence of complete regeneration. Ideally, new functional cardiomyocytes, endothelial cells, and vascular smooth muscle cells should be formed in the necrotic area of the damaged heart. Here, damaged hearts were 9.8% shorter and weighed 14% less than sham controls. In addition, the resection border contained a massive fibrotic scar mainly composed of nonmyocytes and collagen disposition. Furthermore, there was a substantial reduction in the number of proliferating cardiomyocytes in AR hearts. Our results thus question the usefulness of the AR model for identifying molecular mechanisms underlying regeneration of the adult heart after damage.

"The preadipocyte factor" DLK1 marks adult mouse adipose tissue residing vascular cells that lack in vitro adipogenic differentiation potential.

Delta-like 1 (Dlk1) is expressed in 3T3-L1 preadipocytes and has frequently been referred to as "the" preadipocyte marker, yet the phenotype of DLK1(+) cells in adipose tissue remains undetermined. Herein, we demonstrate that DLK1(+) cells encompass around 1-2% of the adult mouse adipose stromal vascular fraction (SVF). Unexpectedly, the DLK1(+)SVF population was enriched for cells expressing genes generally ascribed to the vascular lineage and did not possess any adipogenic differentiation potential in vitro. Instead, DLK1(+) cells comprised an immediate ability for cobblestone formation, generation of tube-like structures on matrigel, and uptake of Acetylated Low Density-Lipoprotein, all characteristics of endothelial cells. We therefore suggest that DLK1(+)SVF cells are of a vascular origin and not them-selves committed preadipocytes as assumed hitherto.