Wounding the stroma: Docetaxel's role in dormant breast cancer escape.

The mechanistic underpinnings of breast cancer recurrence following periods of dormancy are largely undetermined. A new study in PLOS Biology reveals that docetaxel-induced injury of tumour stromal cells stimulates the release of cytokines that support dormancy escape of breast cancer cells.

Lactate Enhances Mouse ES Cell Differentiation Toward XEN Cells In Vitro.

Metabolism plays a crucial role for cell survival and function; however, recent evidence has implicated it in regulating embryonic development. In the embryo, the inner cell mass undergoes orchestrated cellular divisions resulting in the formation of pluripotent epiblast stem cells and primitive endoderm cells. However, both lineages can be captured in vitro as embryonic stem (ES) cells and extraembryonic endoderm (XEN) cells. Concomitantly, changes in the metabolic profile occurs during development, and are well documented in the embryonic lineages. However, a comprehensive multi-omic analysis of these features in XEN cells remains lacking. We observed that mouse XEN cells exhibited high sensitivity to glycolytic inhibition in addition to maintaining elevated intra- and extracellular lactate levels in vitro. Extraembryonic endoderm cells maintain high lactate levels by increased LDHA activity, and re-routing pyruvate away from the mitochondria resulting in reduced mitochondrial activity due to disruptions in electron transport chain stoichiometry. Importantly, exogenous lactate supplementation or promoting intracellular lactate accumulation enhances XEN differentiation in vitro. These results highlight how lactate contributes to XEN differentiation in vitro and may serve to enhance reprogramming efficiency of cells used for regenerative medicine.

Characterization of a Vimentinhigh /Nestinhigh proteome and tissue regenerative secretome generated by human pancreas-derived mesenchymal stromal cells.

Multipotent/mesenchymal stromal cells (MSCs) exist within a variety of postnatal tissues; however, global proteomic analyses comparing tissue-specific MSC are limited. Using human bone marrow (BM)-derived MSCs as a gold standard, we used label-free mass spectrometry and functional assays to characterize the proteome, secretome, and corresponding function of human pancreas-derived MSCs (Panc-MSCs) with a classical phenotype (CD90+/CD73+/CD105+/CD45-/CD31-). Both MSC subtypes expressed mesenchymal markers vimentin, α-SMA, and STRO-1; however, expression of nestin was increased in Panc-MSCs. Accordingly, these Vimentinhigh /Nestinhigh cells were isolated from fresh human pancreatic islet and non-islet tissues. Next, we identified expression of >60 CD markers shared between Panc-MSCs and BM-MSCs, including validated expression of CD14. An additional 19 CD markers were differentially expressed, including reduced pericyte-marker CD146 expression on Panc-MSCs. Panc-MSCs also showed reduced expression of proteins involved in lipid and retinoid metabolism. Accordingly, Panc-MSCs showed restricted responses to adipogenic stimuli in vitro, although both MSC types demonstrated trilineage differentiation. In contrast, Panc-MSCs demonstrated accelerated growth kinetics and competency to pro-neurogenic stimuli in vitro. The secretome of Panc-MSCs was highly enriched for proteins associated with vascular development, wound healing and chemotaxis. Similar to BM-MSCs, Panc-MSCs conditioned media augmented endothelial cell survival, proliferation, and tubule formation in vitro. Importantly, the secretome of both MSC types was capable of stimulating chemotactic infiltration of murine endothelial cells in vivo and reduced hyperglycemia in STZ-treated mice following intrapancreatic injection. Overall, this study provides foundational knowledge to develop Panc-MSCs as a unique MSC subtype with functional properties beneficial in regenerative medicine for diabetes and vascular disease.

Characterization of ovarian cancer-derived extracellular vesicles by surface-enhanced Raman spectroscopy.

Ovarian cancer is the most lethal gynecological malignancy, owing to the fact that most cases are diagnosed at a late stage. To improve prognosis and reduce mortality, we must develop methods for the early diagnosis of ovarian cancer. A step towards early and non-invasive cancer diagnosis is through the utilization of extracellular vesicles (EVs), which are nanoscale, membrane-bound vesicles that contain proteins and genetic material reflective of their parent cell. Thus, EVs secreted by cancer cells can be thought of as cancer biomarkers. In this paper, we present gold nanohole arrays for the capture of ovarian cancer (OvCa)-derived EVs and their characterization by surface-enhanced Raman spectroscopy (SERS). For the first time, we have characterized EVs isolated from two established OvCa cell lines (OV-90, OVCAR3), two primary OvCa cell lines (EOC6, EOC18), and one human immortalized ovarian surface epithelial cell line (hIOSE) by SERS. We subsequently determined their main compositional differences by principal component analysis and were able to discriminate the groups by a logistic regression-based machine learning method with ∼99% accuracy, sensitivity, and specificity. The results presented here are a great step towards quick, facile, and non-invasive cancer diagnosis.

Characterization of extracellular vesicles derived from mesenchymal stromal cells by surface-enhanced Raman spectroscopy.

Extracellular vesicles (EVs) are secreted by all cells into bodily fluids and play an important role in intercellular communication through the transfer of proteins and RNA. There is evidence that EVs specifically released from mesenchymal stromal cells (MSCs) are potent cell-free regenerative agents. However, for MSC EVs to be used in therapeutic practices, there must be a standardized and reproducible method for their characterization. The detection and characterization of EVs are a challenge due to their nanoscale size as well as their molecular heterogeneity. To address this challenge, we have fabricated gold nanohole arrays of varying sizes and shapes by electron beam lithography. These platforms have the dual purpose of trapping single EVs and enhancing their vibrational signature in surface-enhanced Raman spectroscopy (SERS). In this paper, we report SERS spectra for MSC EVs derived from pancreatic tissue (Panc-MSC) and bone marrow (BM-MSC). Using principal component analysis (PCA), we determined that the main compositional differences between these two groups are found at 1236, 761, and 1528 cm-1, corresponding to amide III, tryptophan, and an in-plane -C=C- vibration, respectively. We additionally explored several machine learning approaches to distinguish between BM- and Panc-MSC EVs and achieved 89 % accuracy, 89 % sensitivity, and 88 % specificity using logistic regression.

Inhibition of Aldehyde Dehydrogenase-Activity Expands Multipotent Myeloid Progenitor Cells with Vascular Regenerative Function.

Blood-derived progenitor cell transplantation holds potential for the treatment of severe vascular diseases. Human umbilical cord blood (UCB)-derived hematopoietic progenitor cells purified using high aldehyde dehydrogenase (ALDHhi ) activity demonstrate pro-angiogenic functions following intramuscular (i.m.) transplantation into immunodeficient mice with hind-limb ischemia. Unfortunately, UCB ALDHhi cells are rare and prolonged ex vivo expansion leads to loss of high ALDH-activity and diminished vascular regenerative function. ALDH-activity generates retinoic acid, a potent driver of hematopoietic differentiation, creating a paradoxical challenge to expand UCB ALDHhi cells while limiting differentiation and retaining pro-angiogenic functions. We investigated whether inhibition of ALDH-activity during ex vivo expansion of UCB ALDHhi cells would prevent differentiation and expand progeny that retained pro-angiogenic functions after transplantation into non-obese diabetic/severe combined immunodeficient mice with femoral artery ligation-induced unilateral hind-limb ischemia. Human UCB ALDHhi cells were cultured under serum-free conditions for 9 days, with or without the reversible ALDH-inhibitor, diethylaminobenzaldehyde (DEAB). Although total cell numbers were increased >70-fold, the frequency of cells that retained ALDHhi /CD34+ phenotype was significantly diminished under basal conditions. In contrast, DEAB-inhibition increased total ALDHhi /CD34+ cell number by ≥10-fold, reduced differentiation marker (CD38) expression, and enhanced the retention of multipotent colony-forming cells in vitro. Proteomic analysis revealed that DEAB-treated cells upregulated anti-apoptotic protein expression and diminished production of proteins implicated with megakaryocyte differentiation. The i.m. transplantation of DEAB-treated cells into mice with hind-limb ischemia stimulated endothelial cell proliferation and augmented recovery of hind-limb perfusion. DEAB-inhibition of ALDH-activity delayed hematopoietic differentiation and expanded multipotent myeloid cells that accelerated vascular regeneration following i.m. transplantation in vivo. Stem Cells 2018;36:723-736.

High Aldehyde Dehydrogenase Activity Identifies a Subset of Human Mesenchymal Stromal Cells with Vascular Regenerative Potential.

During culture expansion, multipotent mesenchymal stromal cells (MSCs) differentially express aldehyde dehydrogenase (ALDH), an intracellular detoxification enzyme that protects long-lived cells against oxidative stress. Thus, MSC selection based on ALDH-activity may be used to reduce heterogeneity and distinguish MSC subsets with improved regenerative potency. After expansion of human bone marrow-derived MSCs, cell progeny was purified based on low versus high ALDH-activity (ALDHhi ) by fluorescence-activated cell sorting, and each subset was compared for multipotent stromal and provascular regenerative functions. Both ALDHl ° and ALDHhi MSC subsets demonstrated similar expression of stromal cell (>95% CD73+ , CD90+ , CD105+ ) and pericyte (>95% CD146+ ) surface markers and showed multipotent differentiation into bone, cartilage, and adipose cells in vitro. Conditioned media (CDM) generated by ALDHhi MSCs demonstrated a potent proliferative and prosurvival effect on human microvascular endothelial cells (HMVECs) under serum-free conditions and augmented HMVEC tube-forming capacity in growth factor-reduced matrices. After subcutaneous transplantation within directed in vivo angiogenesis assay implants into immunodeficient mice, ALDHhi MSC or CDM produced by ALDHhi MSC significantly augmented murine vascular cell recruitment and perfused vessel infiltration compared with ALDHl ° MSC. Although both subsets demonstrated strikingly similar mRNA expression patterns, quantitative proteomic analyses performed on subset-specific CDM revealed the ALDHhi MSC subset uniquely secreted multiple proangiogenic cytokines (vascular endothelial growth factor beta, platelet derived growth factor alpha, and angiogenin) and actively produced multiple factors with chemoattractant (transforming growth factor-ß, C-X-C motif chemokine ligand 1, 2, and 3 (GRO), C-C motif chemokine ligand 5 (RANTES), monocyte chemotactic protein 1 (MCP-1), interleukin [IL]-6, IL-8) and matrix-modifying functions (tissue inhibitor of metalloprotinase 1 & 2 (TIMP1/2)). Collectively, MSCs selected for ALDHhi demonstrated enhanced proangiogenic secretory functions and represent a purified MSC subset amenable for vascular regenerative applications. Stem Cells 2017;35:1542-1553.

Expanded Hematopoietic Progenitor Cells Reselected for High Aldehyde Dehydrogenase Activity Demonstrate Islet Regenerative Functions.

Human umbilical cord blood (UCB) hematopoietic progenitor cells (HPC) purified for high aldehyde dehydrogenase activity (ALDH(hi) ) stimulate islet regeneration after transplantation into mice with streptozotocin-induced ß cell deletion. However, ALDH(hi) cells represent a rare progenitor subset and widespread use of UCB ALDH(hi) cells to stimulate islet regeneration will require progenitor cell expansion without loss of islet regenerative functions. Here we demonstrate that prospectively purified UCB ALDH(hi) cells expand efficiently under serum-free, xeno-free conditions with minimal growth factor supplementation. Consistent with the concept that ALDH-activity is decreased as progenitor cells differentiate, kinetic analyses over 9 days revealed the frequency of ALDH(hi) cells diminished as culture time progressed such that total ALDH(hi) cell number was maximal (increased 3-fold) at day 6. Subsequently, day 6 expanded cells (bulk cells) were sorted after culture to reselect differentiated progeny with low ALDH-activity (ALDH(lo) subset) from less differentiated progeny with high ALDH-activity (ALDH(hi) subset). The ALDH(hi) subset retained primitive cell surface marker coexpression (32.0% ± 7.0% CD34(+) /CD38(-) cells, 37.0% ± 6.9% CD34(+) /CD133(+) cells), and demonstrated increased hematopoietic colony forming cell function compared with the ALDH(lo) subset. Notably, bulk cells or ALDH(lo) cells did not possess the functional capacity to lower hyperglycemia after transplantation into streptozotocin-treated NOD/SCID mice. However, transplantation of the repurified ALDH(hi) subset significantly reduced hyperglycemia, improved glucose tolerance, and increased islet-associated cell proliferation and capillary formation. Thus, expansion and delivery of reselected UCB cells that retain high ALDH-activity after short-term culture represents an improved strategy for the development of cellular therapies to enhance islet regeneration in situ.

Association between omega-3 fatty acids and serum prostate-specific antigen.

We examined the association between omega-3 fatty acids (O3FAs) and prostate-specific antigen (PSA) in a cross-sectional analysis of 6219 men examined at the Cooper Clinic from 2009 to 2013. We assayed O3FAs from red blood cell membranes and measured PSA levels in study participants. Multiple logistic regression was used to examine the association between O3FAs and PSA. The mean age of study participants was 55.5 years (SD = 9.8) with a mean PSA level of 1.31 ng/mL (SD = 1.5). Unadjusted analyses indicated that there was a slight, direct association with PSA and each of the O3FAs tested. However, after adjusting for age and body mass index (BMI), the associations were reversed but nonsignificant [odds ratio (OR) for PSA > 4 ng/mL: total omega-3 OR = 0.98 per each 1% of total fatty acids, 95% confidence interval (CI) = 0.93-1.03; docosahexaenoic acid OR = 1.01, 95% CI = 0.92-1.11; omega-3 index OR = 0.99, 95% CI = 0.93-1.05). Similar results were obtained after age and BMI adjustment when the omega-3 index was divided into undesirable (0.01-3.99%), intermediate (4.0-7.99%), and desirable ranges ( ≥ 8.0%). Given that the study had >80% power to detect an odds ratio <0.9 or >1.1, we conclude that associations between O3FAs and PSA levels are either nonexistent or quite weak in the population that this healthy sample represents.

Identifying clinical phenotypes of frontotemporal dementia in post-9/11 era veterans using natural language processing.

Introduction: Frontotemporal dementia (FTD) encompasses a clinically and pathologically diverse group of neurodegenerative disorders, yet little work has quantified the unique phenotypic clinical presentations of FTD among post-9/11 era veterans. To identify phenotypes of FTD using natural language processing (NLP) aided medical chart reviews of post-9/11 era U.S. military Veterans diagnosed with FTD in Veterans Health Administration care. Methods: A medical record chart review of clinician/provider notes was conducted using a Natural Language Processing (NLP) tool, which extracted features related to cognitive dysfunction. NLP features were further organized into seven Research Domain Criteria Initiative (RDoC) domains, which were clustered to identify distinct phenotypes. Results: Veterans with FTD were more likely to have notes that reflected the RDoC domains, with cognitive and positive valence domains showing the greatest difference across groups. Clustering of domains identified three symptom phenotypes agnostic to time of an individual having FTD, categorized as Low (16.4%), Moderate (69.2%), and High (14.5%) distress. Comparison across distress groups showed significant differences in physical and psychological characteristics, particularly prior history of head injury, insomnia, cardiac issues, anxiety, and alcohol misuse. The clustering result within the FTD group demonstrated a phenotype variant that exhibited a combination of language and behavioral symptoms. This phenotype presented with manifestations indicative of both language-related impairments and behavioral changes, showcasing the coexistence of features from both domains within the same individual. Discussion: This study suggests FTD also presents across a continuum of severity and symptom distress, both within and across variants. The intensity of distress evident in clinical notes tends to cluster with more co-occurring conditions. This examination of phenotypic heterogeneity in clinical notes indicates that sensitivity to FTD diagnosis may be correlated to overall symptom distress, and future work incorporating NLP and phenotyping may help promote strategies for early detection of FTD.

Tandem SERS and MS/MS Profiling of Plasma Extracellular Vesicles for Early Ovarian Cancer Biomarker Discovery.

Extracellular vesicles (EVs) are vectors of biomolecular cargo that play essential roles in intercellular communication across a range of cells. Protein, lipid, and nucleic acid cargo harbored within EVs may serve as biomarkers at all stages of disease; however, the choice of methodology may challenge the specificity and reproducibility of discovery. To address these challenges, the integration of rigorous EV purification methods, cutting-edge spectroscopic technologies, and data analysis are critical to uncover diagnostic signatures of disease. Herein, we demonstrate an EV isolation and analysis pipeline using surface-enhanced Raman spectroscopy (SERS) and mass spectrometry (MS) techniques on plasma samples obtained from umbilical cord blood, healthy donor (HD) plasma, and plasma from women with early stage high-grade serous carcinoma (HGSC). Plasma EVs were purified by size exclusion chromatography and analyzed by surface-enhanced Raman spectroscopy (SERS), mass spectrometry (MS), and atomic force microscopy. After determining the fraction of highest EV purity, SERS and MS were used to characterize EVs from HDs, pooled donors with noncancerous gynecological ailments (n = 6), and donors with early stage [FIGO (I/II)] with HGSC. SERS spectra were subjected to different machine learning algorithms such as PCA, logistic regression, support vector machine, naïve Bayes, random forest, neural network, and k nearest neighbors to differentiate healthy, benign, and HGSC EVs. Collectively, we demonstrate a reproducible workflow with the potential to serve as a diagnostic platform for HGSC.

Targeted proteomics of plasma extracellular vesicles uncovers MUC1 as combinatorial biomarker for the early detection of high-grade serous ovarian cancer.

BACKGROUND: The five-year prognosis for patients with late-stage high-grade serous carcinoma (HGSC) remains dismal, underscoring the critical need for identifying early-stage biomarkers. This study explores the potential of extracellular vesicles (EVs) circulating in blood, which are believed to harbor proteomic cargo reflective of the HGSC microenvironment, as a source for biomarker discovery. RESULTS: We conducted a comprehensive proteomic profiling of EVs isolated from blood plasma, ascites, and cell lines of patients, employing both data-dependent (DDA) and data-independent acquisition (DIA) methods to construct a spectral library tailored for targeted proteomics. Our investigation aimed at uncovering novel biomarkers for the early detection of HGSC by comparing the proteomic signatures of EVs from women with HGSC to those with benign gynecological conditions. The initial cohort, comprising 19 donors, utilized DDA proteomics for spectral library development. The subsequent cohort, involving 30 HGSC patients and 30 control subjects, employed DIA proteomics for a similar purpose. Support vector machine (SVM) classification was applied in both cohorts to identify combinatorial biomarkers with high specificity and sensitivity (ROC-AUC > 0.90). Notably, MUC1 emerged as a significant biomarker in both cohorts when used in combination with additional biomarkers. Validation through an ELISA assay on a subset of benign (n = 18), Stage I (n = 9), and stage II (n = 9) plasma samples corroborated the diagnostic utility of MUC1 in the early-stage detection of HGSC. CONCLUSIONS: This study highlights the value of EV-based proteomic analysis in the discovery of combinatorial biomarkers for early ovarian cancer detection.

Probing the effects of matrix-derived microcarrier composition on human adipose-derived stromal cells cultured dynamically within spinner flask bioreactors.

Recognizing the cell-instructive capacity of the extracellular matrix (ECM), this study investigated the effects of expanding human adipose-derived stromal cells (hASCs) on ECM-derived microcarriers fabricated from decellularized adipose tissue (DAT) or decellularized cartilage tissue (DCT) within spinner flask bioreactors. Protocols were established for decellularizing porcine auricular cartilage and electrospraying methods were used to generate microcarriers comprised exclusively of DAT or DCT, which were compositionally distinct, but had matching Young's moduli. Both microcarrier types supported hASC attachment and growth over 14 days within a low-shear spinner culture system, with a significantly higher cell density observed on the DCT microcarriers at 7 and 14 days. Irrespective of the ECM source, dynamic culture on the microcarriers altered the expression of genes and proteins associated with cell adhesion and ECM remodeling. Label-free mass spectrometry analysis showed upregulation of proteins associated with cartilage development and ECM in the hASCs expanded on the DCT microcarriers. Based on Luminex analysis, the hASCs expanded on the DCT microcarriers secreted significantly higher levels of IL-8 and PDGFAA, supporting that the ECM source can modulate hASC paracrine factor secretion. Finally, the hASCs expanded on the microcarriers were extracted for analysis of adipogenic and chondrogenic differentiation relative to baseline controls. The microcarrier-cultured hASCs showed enhanced intracellular lipid accumulation at 7 days post-induction of adipogenic differentiation. In the chondrogenic studies, a low level of differentiation was observed in all groups. Future studies are warranted using alternative cell sources with greater chondrogenic potential to further assess the chondro-inductive properties of the DCT microcarriers.

Never in Mitosis Kinase 2 regulation of metabolism is required for neural differentiation.

Wnt and Hh are known signalling pathways involved in neural differentiation and recent work has shown the cell cycle regulator, Never in Mitosis Kinase 2 (Nek2) is able to regulate both pathways. Despite its known function in pathway regulation, few studies have explored Nek2 within embryonic development. The P19 embryonal carcinoma cell model was used to investigate Nek2 and neural differentiation through CRISPR knockout and overexpression studies. Loss of Nek2 reduced cell proliferation in the undifferentiated state and during directed differentiation, while overexpression increased cell proliferation. Despite these changes in proliferation rates, Nek2 deficient cells maintained pluripotency markers after neural induction while Nek2 overexpressing cells lost these markers in the undifferentiated state. Nek2 deficient cells lost the ability to differentiate into both neurons and astrocytes, although Nek2 overexpressing cells enhanced neuron differentiation at the expense of astrocytes. Hh and Wnt signalling were explored, however there was no clear connection between Nek2 and these pathways causing the observed changes to differentiation phenotypes. Mass spectrometry was also used during wildtype and Nek2 knockout cell differentiation and we identified reduced electron transport chain components in the knockout population. Immunoblotting confirmed the loss of these components and additional studies showed cells lacking Nek2 were exclusively glycolytic. Interestingly, hypoxia inducible factor 1α was stabilized in these Nek2 knockout cells despite culturing them under normoxic conditions. Since neural differentiation requires a metabolic switch from glycolysis to oxidative phosphorylation, we propose a mechanism where Nek2 prevents HIF1α stabilization, thereby allowing cells to use oxidative phosphorylation to facilitate neuron and astrocyte differentiation.

Decellularized adipose tissue scaffolds guide hematopoietic differentiation and stimulate vascular regeneration in a hindlimb ischemia model.

Cellular therapies to stimulate therapeutic angiogenesis in individuals with critical limb ischemia (CLI) remain under intense investigation. In this context, the efficacy of cell therapy is dependent on the survival, biodistribution, and pro-angiogenic paracrine signaling of the cells transplanted. Hematopoietic progenitor cells (HPC) purified from human umbilical cord blood using high aldehyde dehydrogenase-activity (ALDHhi cells) and expanded ex vivo, represent a heterogeneous mixture of progenitor cells previously shown to support limb revascularization in mouse models of CLI. The objectives of this study were to investigate the utility of bioscaffolds derived from human decellularized adipose tissue (DAT) to guide the differentiation of seeded HPC in vitro and harness the pro-angiogenic capacity of HPC at the site of ischemia after implantation in vivo. Probing whether the DAT scaffolds altered HPC differentiation, label-free quantitative mass spectrometry and flow cytometric phenotype analyses indicated that culturing the HPC on the DAT scaffolds supported their differentiation towards the pro-angiogenic monocyte/macrophage lineage at the expense of megakaryopoiesis. Moreover, implantation of HPC in DAT scaffolds within a unilateral hindlimb ischemia model in NOD/SCID mice increased cell retention at the site of ischemia relative to intramuscular injection, and accelerated the recovery of limb perfusion, improved functional limb use and augmented CD31+ capillary density when compared to DAT implantation alone or saline-injected controls. Collectively, these data indicate that cell-instructive DAT scaffolds can direct therapeutic HPC differentiation towards the monocyte/macrophage lineage and represent a promising delivery platform for improving the efficacy of cell therapies for CLI.

Accumulation of Good Intentions: How Individual Practice Guidelines Lead to Polypharmacy in the Treatment of Patients with Polytrauma.

Polytrauma clinical triad (PCT) is the comorbid occurrence of traumatic brain injury (TBI), posttraumatic stress disorder (PTSD), and pain after trauma. No clinical practice guidelines for postacute care of patients with PCT currently exist; instead, clinical practice guidelines have been published for the three conditions (TBI, PTSD, and pain) as distinct clinical entities. Using multiple, individual practice guidelines for a patient with PCT may lead to unintended prescription of multiple and potentially adversely interacting medications (ie, polypharmacy). Polypharmacy, especially that which includes central nervous system-acting medications, may lead to overdose, suicidality, and chronic symptomatology. Current individual guidelines for each condition of PCT do not address how to coordinate care for the polytraumatic diagnosis. The purpose of this Practice Management piece is to describe the unintended consequences of polypharmacy in patients with PCT and to discuss mitigation approaches including rational prescribing, nonpharmacologic alternatives, and interdisciplinary coordination.

Ultrafiltration and Injection of Islet Regenerative Stimuli Secreted by Pancreatic Mesenchymal Stromal Cells.

The secretome of mesenchymal stromal cells (MSCs) is enriched for biotherapeutic effectors contained within and independent of extracellular vesicles (EVs) that may support tissue regeneration as an injectable agent. We have demonstrated that the intrapancreatic injection of concentrated conditioned media (CM) produced by bone marrow MSC supports islet regeneration and restored glycemic control in hyperglycemic mice, ultimately providing a platform to elucidate components of the MSC secretome. Herein, we extend these findings using human pancreas-derived MSC (Panc-MSC) as "biofactories" to enrich for tissue regenerative stimuli housed within distinct compartments of the secretome. Specifically, we utilized 100 kDa ultrafiltration as a simple method to debulk protein mass and to enrich for EVs while concentrating the MSC secretome into an injectable volume for preclinical assessments in murine models of blood vessel and islet regeneration. EV enrichment (EV+) was validated using nanoscale flow cytometry and atomic force microscopy, in addition to the detection of classical EV markers CD9, CD81, and CD63 using label-free mass spectrometry. EV+ CM was predominately enriched with mediators of wound healing and epithelial-to-mesenchymal transition that supported functional regeneration in mesenchymal and nonmesenchymal tissues. For example, EV+ CM supported human microvascular endothelial cell tubule formation in vitro and enhanced the recovery of blood perfusion following intramuscular injection in nonobese diabetic/severe combined immunodeficiency mice with unilateral hind limb ischemia. Furthermore, EV+ CM increased islet number and ß cell mass, elevated circulating insulin, and improved glycemic control following intrapancreatic injection in streptozotocin-treated mice. Collectively, this study provides foundational evidence that Panc-MSC, readily propagated from the subculture of human islets, may be utilized for regenerative medicine applications.

Purification and Functional Characterization of CD34-Expressing Cell Subsets Following Ex Vivo Expansion of Umbilical Cord Blood-Derived Endothelial Colony-Forming Cells.

Fluorescent-activated cell sorting (FACS) remains a powerful tool to enrich blood-derived progenitor cells for the establishment of highly proliferative endothelial colony-forming cells (ECFC). Further investigation remains necessary to determine whether the retention of progenitor cell phenotypes after expansion can identify ECFC with enhanced proangiogenic and regenerative functions. This study employed FACS purification to segregate umbilical cord blood-derived ECFC using conserved provascular progenitor cell markers CD34 or aldehyde dehydrogenase (ALDH) activity. ECFC FACS purified for high versus low ALDH activity formed single cell-derived colonies and demonstrated tubule formation in Matrigel at comparable rates. Surprisingly, FACS purification of ECFC for CD34 enriched cells with enhanced colony-forming capabilities and tubule formation within the CD34- population. CD34 expression was enriched on early ECFC populations; however, steady-state expression of CD34 rapidly declined and stabilized on expanded ECFC after serial passage. CD34 expression on ECFC was shown to be cell density dependent and coincided with a loss of progenitor cell characteristics in vitro. Silica-bead surface membrane capture followed by proteomic analysis by label-free liquid chromatography tandem mass spectrometry (LC-MS/MS) identified >100 distinctions (P < 0.05) associated with the plasma membrane of CD34- versus CD34+ ECFC, including a significant enrichment of CD143 (angiotensinogen converting enzyme) on CD34+ cells. Despite an enrichment for traditional endothelial cell markers on the CD34+ ECFC in vitro, implantation of both CD34+ and CD34- ECFC within Matrigel plugs in immunodeficient NOD.SCID mice promoted the formation of vessel-like structures with equivalent integration of human cells at 7 days post-transplantation. Although positive selection of CD34 enriched ECFC establishment before culture, FACS-purified CD34+ ECFC demonstrated reduced colony and tubule formation in vitro, yet demonstrated equivalent vessel formative function in vivo compared to CD34- counterparts. The knowledge will support future studies aiming to identify ECFC subsets with enhanced vessel forming functions for applications of regenerative medicine.

Traditional signs and symptoms commonly attributed to hypogonadism do not correlate with testosterone levels: the Cooper Center Longitudinal Study Experience.

Evidence suggests that substantial testosterone therapy is occurring without checking levels of testosterone, presumably based on the presence of symptoms alone. We sought to explore the relationship between total testosterone level and non-specific symptoms, metabolic abnormalities, and sexual dysfunction associated with hypogonadism. This cross-sectional study included 2994 generally healthy men aged 50-79 years examined at a preventive medicine clinic in Dallas, TX from January 2012 to March 2016. Symptoms of hypogonadism were assessed. Screening morning total testosterone levels were measured and categorized into low (<250 ng/dL), low normal (250-399 ng/dL), and normal (≥400 ng/dL). Multiple logistic regression models were used to test the associations between total testosterone and signs and symptoms of hypogonadism. When considering symptoms and signs of hypogonadism, only decreased libido (OR 1.31, 95% CI 1.00 to 1.70), fasting glucose ≥100 mg/dL (OR 1.47, CI 1.15 to 1.88), and hemoglobin A1c over 6% (OR 1.47, 95% CI 1.06 to 2.03) were associated with increased odds of low testosterone after adjustment for age, body mass index, and cardiorespiratory fitness. Testosterone levels were not associated with fatigue, depression, or erectile dysfunction in our study (p>0.6). In this preventive medicine cohort, symptoms commonly attributed to testosterone deficiency were not associated with low total testosterone levels.

Vascular Organoids: Are We Entering a New Area of Cardiometabolic Research?

Recently in Nature, Wimmer et al. (2019) reported on the development of a human organoid model of vascular development recapitulating vascular pathology during type 2 diabetes. Integration of these organoids into the vasculature of immunodeficient mice may provide cardiometabolic researchers with a "personalized" platform for novel therapeutic discovery.