We established a functional adipose organoid model system for human adipose stem/progenitor cells (ASCs) isolated from white adipose tissue (WAT). ASCs were forced to self-aggregate by a hanging-drop technique. Afterwards, spheroids were transferred into agar-coated cell culture dishes to avoid plastic-adherence and dis-aggregation. Adipocyte differentiation was induced by an adipogenic hormone cocktail. Morphometric analysis revealed a significant increase in organoid size in the course of adipogenesis until d 18. Whole mount staining of organoids using specific lipophilic dyes showed large multi- and unilocular fat deposits in differentiated cells indicating highly efficient differentiation of ASCs into mature adipocytes. Moreover, we found a strong induction of the expression of key adipogenesis and adipocyte markers (CCAAT/enhancer-binding protein (C/EBP) β, peroxisome proliferator-activated receptor (PPAR) γ, fatty acid-binding protein 4 (FABP4), adiponectin) during adipose organoid formation. Secreted adiponectin was detected in the cell culture supernatant, underscoring the physiological relevance of mature adipocytes in the organoid model. Moreover, colony formation assays of collagenase-digested organoids revealed the maintenance of a significant fraction of ASCs within newly formed organoids. In conclusion, we provide a reliable and highly efficient WAT organoid model, which enables accurate analysis of cellular and molecular markers of adipogenic differentiation and adipocyte physiology.

We explore the status of quiescence, stemness and adipogenic differentiation capacity in adipose stem/progenitor cells (ASCs) ex vivo, immediately after isolation from human subcutaneous white adipose tissue, by sorting the stromal vascular fraction into cell-surface DLK1⁺/CD34⁻, DLK1⁺/CD34^dim and DLK1⁻/CD34⁺ cells. We demonstrate that DLK1⁻/CD34⁺ cells, the only population exhibiting proliferative and adipogenic capacity, express ex vivo the bonafide quiescence markers p21^Cip1, p27^Kip1 and p57^Kip2 but neither proliferation markers nor the senescence marker p16^Ink4a. The pluripotency markers NANOG, SOX2 and OCT4 are barely detectable in ex vivo ASCs while the somatic stemness factors, c-MYC and KLF4 and the early adipogenic factor C/EBPβ are highly expressed. Further sorting of ASCs into DLK1⁻/CD34⁺/CD24⁻ and DLK1⁻/CD34⁺/CD24⁺ fractions shows that KLF4 and c-MYC are higher expressed in DLK1⁻/CD34⁺/CD24⁺ cells correlating with higher colony formation capacity and considerably lower adipogenic activity. Proliferation capacity is similar in both populations. Next, we show that ASCs routinely isolated by plastic-adherence are DLK1⁻/CD34⁺/CD24⁺. Intriguingly, CD24 knock-down in these cells reduces proliferation and adipogenesis. In conclusion, DLK1⁻/CD34⁺ ASCs in human sWAT exist in a quiescent state, express high levels of somatic stemness factors and the early adipogenic transcription factor C/EBPβ but senescence and pluripotency markers are barely detectable. Moreover, our data indicate that CD24 is necessary for adequate ASC proliferation and adipogenesis and that stemness is higher and adipogenic capacity lower in DLK1⁻/CD34⁺/CD24⁺ relative to DLK1⁻/CD34⁺/CD24⁻ subpopulations.

Background: The pedicled pectoralis major muscle flap (PMMF) is a well established flap for fistula prophylaxis after salvage laryngectomy. To reduce donor site morbidity, we established a modified muscle-sparing harvesting technique. We herein investigate postoperative shoulder function and health-related quality of life (HRQOL). Methods: A chart review of patients receiving the modified muscle-sparing pectoralis major muscle flap between 2013–2020 was performed. Nineteen patients (male = 18, female = 1) were potentially eligible and six male patients were ultimately enrolled. Postoperative shoulder function was assessed on both sides (flap side versus non-flap side) using the Constant Murley Score and the Bak criteria. Health-related quality of life was assessed with the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire in cancer patients (EORTC QLQ-C30) and head and neck cancer patients (EORTC H&N35). Results: No Constant Murley Score subscale was statistically significant (p ≥ 0.180). Bak criteria was overall rated “Good“. Solely upper extremity adduction force was significantly altered on the flap side (p = 0.039). Median EORTC QLQ-C30 score was 82.2 (IQR 11.1) on the functional scale and 10.3 (IQR 2.6) on the symptomatic scale. Median quality of life score was 75.0 (IQR 33.3) and median EORTC QLQ-H&N35 was 20.6 (IQR 9.8). Conclusions: Postoperative shoulder function after modified muscle-sparing pectoralis major muscle flap surgery is comparable to function of the healthy side with a significant deficiency in adduction force not compromising daily life in this small study cohort.

Misalignment of physiological circadian rhythms promotes obesity which is characterized by white adipose tissue (WAT) expansion. Differentiation of Adipose stem/progenitor cells (ASCs) contributes to WAT increase but the importance of the cellular clock in this process is incompletely understood. In the present study, we reveal the role of the circadian transcription factor Aryl hydrocarbon receptor nuclear translocator-like 2 (ARNTL2) in human ASCs, isolated from subcutaneous (s)WAT samples of patients undergoing routine elective plastic abdominal surgery. We show that circadian synchronization by serum-shock or stimulation with adipogenic stimuli leads to a different expression pattern of ARNTL2 relative to its well-studied paralogue ARNTL1. We demonstrate that ARNTL2 mRNA is downregulated in ASCs upon weight-loss (WL) whereas ARNTL2 protein is rapidly induced in the course of adipogenic differentiation and highly abundant in adipocytes. ARNTL2 protein is maintained in ASCs cooperatively by mechanistic Target of Rapamycin (mTOR) and Mitogen-activated Protein Kinase (MAPK) signalling pathways while ARNTL2 functions as an inhibitor on both circuits, leading to a feedback mechanism. Consistently, ectopic overexpression of ARNTL2 repressed adipogenesis by facilitating the degradation of ARNTL1, inhibition of Kruppel-Like Factor 15 (KLF15) gene expression and down-regulation of the MAPK-CCAAT/enhancer-binding protein β (C/EBPβ) axis. Western blot analysis of sWAT samples from normal-weight, obese and WL donors revealed that ARNTL2 protein was solely elevated by WL compared to ARNTL1 which underscores unique functions of both transcription factors. In conclusion, our study reveals ARNTL2 to be a WL-regulated inhibitor of adipogenesis which might provide opportunities to develop strategies to ameliorate obesity.

Background: Pectoralis major flap reconstruction is often associated with large unappealing scars in head-neck surgery. We recently established an alternative harvesting technique that improves aesthetic outcome. Objectives: The objective of this study was to demonstrate a modified surgical technique that harvests the pectoralis major muscle as an island flap and focuses on minimizing incision lines and applying a muscle-sparing approach. Methods: A retrospective analysis covering the period 2008–2018 was conducted. Patients who underwent pectoralis major island flap reconstruction for fistula prophylaxis after salvage laryngectomy at the Medical University of Innsbruck were included. Flap harvesting was performed subcutaneously using two small incision lines, thereby sparing the clavicular and upper sternocostal aspects of the pectoralis muscle. Results: Twenty-three patients with squamous cell carcinoma of the larynx (n = 19) or pharynx (n = 4) underwent salvage laryngectomy and consecutive reconstruction using a pectoralis major island flap and our muscle-sparing technique. Mean overall operation time was 147 ± 48.6 min. The majority (70%) of patients had an uneventful course of recovery and showed satisfying aesthetic results and low donor site morbidity as compared to traditional harvesting techniques. Six (26%) patients had major complications that required surgical revision. Three (13%) cases were complicated with fistulas. Conclusion: The pectoralis major island flap is shown to be reliable in otolaryngeal surgery. This surgical technique minimizes scarring and preserves thoracic wall architecture by taking a less invasive approach, thereby improving aesthetic outcome and reducing overall patient morbidity.

The CRISPR/Cas9 system is a powerful tool to generate a specific loss-of-function phenotype by gene knockout (KO). However, this approach is challenging in primary human cells. In this technical report, we present a reliable protocol to achieve a functional KO in the genome of human adipose stem/progenitor cells (ASCs). Using Sprouty1 (SPRY1) as a model target gene for a CRISPR/Cas9 mediated KO, we particularize the procedure including the selection of the CRISPR/Cas9 target sequences and the employment of appropriate lentiviral vectors to obtain a functional gene KO. The efficiency of CRISPR/Cas9 to mutate the SPRY1 gene is determined by a PCR-based mutation detection assay and sequence analysis. Effects on mRNA and protein levels are studied by RT-qPCR and Western blotting. In addition, we demonstrate that CRISPR/Cas9 mediated SPRY1 KO and gene silencing by shRNA are similarly effective to deplete the Sprouty1 protein and to inhibit adipogenic differentiation. In summary, we show a reliable approach to achieve a gene KO in human ASCs, which could also apply to other primary cell types. Abbreviations: ASC: Adipogenic Stem/Progenitor Cell; Cas: CRISPR-associated system; CRISPR: Clustered Regularly Interspaced Palindromic Repeat; gDNA: Genomic DNA; GOI: Gene of interest; gRNA: Guide RNA; NHEJ: Non-homologous end joining; Indel: Insertion/Deletion; PAM: Protospacer adjacent motif; sWAT: Subcutaneous white adipose tissue; TIDE: Tracking of indels by decomposition