p57Kip2 regulates embryonic blood stem cells by controlling sympathoadrenal progenitor expansion.

Hematopoietic stem cells (HSCs) are of major clinical importance, and finding methods for their in vitro generation is a prime research focus. We show here that the cell cycle inhibitor p57Kip2/Cdkn1c limits the number of emerging HSCs by restricting the size of the sympathetic nervous system (SNS) and the amount of HSC-supportive catecholamines secreted by these cells. This regulation occurs at the SNS progenitor level and is in contrast to the cell-intrinsic function of p57Kip2 in maintaining adult HSCs, highlighting profound differences in cell cycle requirements of adult HSCs compared with their embryonic counterparts. Furthermore, this effect is specific to the aorta-gonad-mesonephros (AGM) region and shows that the AGM is the main contributor to early fetal liver colonization, as early fetal liver HSC numbers are equally affected. Using a range of antagonists in vivo, we show a requirement for intact ß2-adrenergic signaling for SNS-dependent HSC expansion. To gain further molecular insights, we have generated a single-cell RNA-sequencing data set of all Ngfr+ sympathoadrenal cells around the dorsal aorta to dissect their differentiation pathway. Importantly, this not only defined the relevant p57Kip2-expressing SNS progenitor stage but also revealed that some neural crest cells, upon arrival at the aorta, are able to take an alternative differentiation pathway, giving rise to a subset of ventrally restricted mesenchymal cells that express important HSC-supportive factors. Neural crest cells thus appear to contribute to the AGM HSC niche via 2 different mechanisms: SNS-mediated catecholamine secretion and HSC-supportive mesenchymal cell production.

Concise review: From greenhouse to garden: the changing soil of the hematopoietic stem cell microenvironment during development.

The hematopoietic system has been intensely studied for many decades. For this reason, it has become the best understood stem cell-derived system that serves as a paradigm for stem cell biology and has found numerous applications in the clinics. While a lot of progress has recently been made in describing the bone marrow components that maintain and control blood stem cell function in the adult, very little is currently known about the regulatory microenvironment in which the first adult-repopulating hematopoietic stem cells are formed during development. Knowledge of these processes is crucial for understanding the basic regulation of hematopoietic stem cell production and behavior and to allow their in vitro expansion and generation from embryonic stem cells or iPS cells for clinical and research purposes. This review summarizes the recent advances that have been made in defining the cellular components, as well as the soluble and physical factors, that are part of the niche involved in regulating hematopoietic stem cell generation in the embryo. The findings are compared with what is known about the adult bone marrow niche to find common pathways for stem cell regulation, but also to highlight processes uniquely required for de novo hematopoietic stem cell generation, as these are the conditions that will need to be recreated for the successful production of blood stem cells in culture.

The Extended Chest Wall Perforator Flap: Expanding the Indication for Partial Breast Reconstruction.

Background: The intercostal artery perforator flap has traditionally been used to reconstruct small or moderate-sized single defects in the lateral or lower medial breast during breast-conserving surgery. We report a modification of the intercostal artery perforator flap that allows for reconstruction of larger breast tumors than previously described flap designs. Methods: A retrospective study of breast cancer patients undergoing breast-conserving surgery and immediate partial breast reconstruction with an extended chest wall perforator flap. Primary outcomes were successful tumor excision, adequate radial margins, postoperative complications, and delays to adjuvant radiotherapy. Results: Thirty patients were included. Mean radiological tumor size was 27 mm (11-56 mm) and excision volume, 123 cm3 (18-255 cm3). All tumors had satisfactory excision margins, and no patient required further surgery for re-excision. In the early postoperative period, one patient required radiological drainage of seroma, and one returned to theater for debridement of fat necrosis affecting the flap. Ten other patients were managed on an outpatient basis for minor wound complications. All patients were followed up annually for 5 years. No patients had a delay to adjuvant treatment or required revisional procedures for cosmesis. Conclusions: The modified chest wall perforator flap allows for breast conservation for larger tumors from all quadrants of the breast, including centrally located tumors and reconstruction of the axillary defect following lymph node clearance. The length of the flap allows for the use of multiple perforators in the pedicle area and freedom of the flap to reach the defects. This can be performed with low morbidity and no delay to adjuvant radiotherapy.

Dlk1 is a negative regulator of emerging hematopoietic stem and progenitor cells.

The first mouse adult-repopulating hematopoietic stem cells emerge in the aorta-gonad-mesonephros region at embryonic day (E) 10.5. Their numbers in this region increase thereafter and begin to decline at E12.5, thus pointing to the possible existence of both positive and negative regulators of emerging hematopoietic stem cells. Our recent expression analysis of the aorta-gonad-mesonephros region showed that the Delta-like homologue 1 (Dlk1) gene is up-regulated in the region of the aorta-gonad-mesonephros where hematopoietic stem cells are preferentially located. To analyze its function, we studied Dlk1 expression in wild-type and hematopoietic stem cell-deficient embryos and determined hematopoietic stem and progenitor cell activity in Dlk1 knockout and overexpressing mice. Its role in hematopoietic support was studied in co-culture experiments using stromal cell lines that express varying levels of Dlk1. We show here that Dlk1 is expressed in the smooth muscle layer of the dorsal aorta and the ventral sub-aortic mesenchyme, where its expression is dependent on the hematopoietic transcription factor Runx1. We further demonstrate that Dlk1 has a negative impact on hematopoietic stem and progenitor cell activity in the aorta-gonad-mesonephros region in vivo, which is recapitulated in co-cultures of hematopoietic stem cells on stromal cells that express varying levels of Dlk1. This negative effect of Dlk1 on hematopoietic stem and progenitor cell activity requires the membrane-bound form of the protein and cannot be recapitulated by soluble Dlk1. Together, these data suggest that Dlk1 expression by cells of the aorta-gonad-mesonephros hematopoietic microenvironment limits hematopoietic stem cell expansion and is, to our knowledge, the first description of such a negative regulator in this tissue.

Simple oncoplastic breast defect closure improves long-term cosmetic outcome of breast conserving surgery for breast cancer: A randomised controlled trial.

INTRODUCTION: Breast conserving surgery (BCS) is associated with unsatisfactory cosmetic outcomes in up to 30% of patients, carrying psychological and quality-of-life implications. This study compares long-term cosmetic outcomes after BCS for breast cancer with v without simple oncoplastic defect closure. METHODS: A randomised controlled trial was performed, recruiting patients who underwent BCS over four years and randomising to the "reshaping" group (closure of excision defect with mobilised breast tissue; n = 124) and to the "control" group (no attempt at defect closure; n = 109). The estimated excision volume (EEV) was <20% of breast volume (BV) in both groups. Photography and breast retraction assessment (BRA) were recorded preoperatively. Cosmetic outcomes were blindly assessed annually for five years by BRA, panel assessment of patients, and body image questionnaire (BIQ). RESULTS: There were no significant differences between the reshaping and control groups in mean age (52.4 v 53.0; p = 0.63), body mass index (27.8 v 27.7; p = 0.80), margin re-excision (9 v 9; p = 0.78), mean BV (562.5 v 590.3 cc; p = 0.56), mean EEV (54.6 v 60.1 cc; p = 0.14), mean EEV/BV ratio (11.2 v 11.0; p = 0.84), or mean specimen weight (52.1 v 57.7 g; p = 0.24). Reshaping group patients had significantly better outcomes compared to control group patients in terms of mean BRA (0.9 v 2.8; p < 0.0001), achieving a score of "good" or "excellent" by panel assessment at 5 years (75.8% v 48%, p < 0.0001), body image questionnaire top score at 5 years (66.9% v 35.8%; p = 0.0001). CONCLUSIONS: Simple oncoplastic closure of defects after breast-conserving surgery improves long-term objective and subjective cosmetic outcomes.

Genome-wide screening identifies Polycomb repressive complex 1.3 as an essential regulator of human naïve pluripotent cell reprogramming.

Uncovering the mechanisms that establish naïve pluripotency in humans is crucial for the future applications of pluripotent stem cells including the production of human blastoids. However, the regulatory pathways that control the establishment of naïve pluripotency by reprogramming are largely unknown. Here, we use genome-wide screening to identify essential regulators as well as major impediments of human primed to naïve pluripotent stem cell reprogramming. We discover that factors essential for cell state change do not typically undergo changes at the level of gene expression but rather are repurposed with new functions. Mechanistically, we establish that the variant Polycomb complex PRC1.3 and PRDM14 jointly repress developmental and gene regulatory factors to ensure naïve cell reprogramming. In addition, small-molecule inhibitors of reprogramming impediments improve naïve cell reprogramming beyond current methods. Collectively, this work defines the principles controlling the establishment of human naïve pluripotency and also provides new insights into mechanisms that destabilize and reconfigure cell identity during cell state transitions.

Assay of advanced glycation endproducts in selected beverages and food by liquid chromatography with tandem mass spectrometric detection.

Food and beverages contain protein glycation adducts--both early-stage adducts and advanced glycation endproducts. We determined the concentrations of glycation adducts in selected food and beverages by liquid chromatography with triple quadrupole mass spectrometric detection. Cola drink contained low concentrations of glycation free adducts, whereas pasteurised and sterilised milk were rich sources of heat-stable glycation adduct residues--Nepsilon-carboxymethyl-lysine and Nepsilon-carboxyethyl-lysine. Laboratory rodent food was a rich source of advanced glycation endproducts. Measurement of glycation adducts in 24 h urine samples of normal and diabetic rats indicated that < 10% of glycation adduct residue consumption was excreted. Induction of diabetes by streptozotocin led to a 2-fold increase in urinary excretion of Nepsilon-carboxymethyl-lysine and a 27-fold increase in urinary excretion of methylglyoxal-derived hydroimidazolone Ndelta-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine - the latter was decreased by high-dose thiamine therapy that also prevented the development of nephropathy. We conclude that cola drinks are a poor source of glycation adduct whereas thermally processed milk is rich in glycation adducts. Dietary glycation adducts residues probably have low bioavailability. Experimental diabetes is associated with a marked increase in exposure to endogenous formation of methylglyoxal-derived hydroimidazolone which is linked to the development of diabetic nephropathy.

Signaling from the sympathetic nervous system regulates hematopoietic stem cell emergence during embryogenesis.

The first adult-repopulating hematopoietic stem cells (HSCs) emerge in the aorta-gonads-mesonephros (AGM) region of the embryo. We have recently identified the transcription factor Gata3 as being upregulated in this tissue specifically at the time of HSC emergence. We now demonstrate that the production of functional and phenotypic HSCs in the AGM is impaired in the absence of Gata3. Furthermore, we show that this effect on HSC generation is secondary to the role of Gata3 in the production of catecholamines, the mediators of the sympathetic nervous system (SNS), thus making these molecules key components of the AGM HSC niche. These findings demonstrate that the recently described functional interplay between the hematopoietic system and the SNS extends to the earliest stages of their codevelopment and highlight the fact that HSC development needs to be viewed in the context of the development of other organs.