Idiopathic Subglottic Stenosis Is Associated With More Frequent and Abnormal Squamous Metaplasia.

OBJECTIVES: Gain insights into the pathophysiology of idiopathic subglottic stenosis (iSGS) by investigating differences in transcriptome of subglottic mucosal tissue between patients with iSGS and controls, and between tracheal and subglottic tissue within patients. METHODS: RNA sequencing was conducted on biopsied mucosal samples collected from subglottic and tracheal (in-patient control) regions in iSGS patients, and from subglottis in controls. The gene expression differences were validated on a protein level by (1) staining the tissue samples obtained from a second cohort of patients and controls; and (2) in vitro functional assays using primary subglottic epithelial cells from both iSGS patients and healthy donors. RESULTS: We found 7 upregulated genes in the subglottic region of iSGS patients relative to both the tracheal mucosa and subglottic region of controls. A gene ontology enrichment analysis found that the epithelial cell differentiation and cornification pathways are significant, involving specifically 3 of the genes: involucrin (IVL), small proline rich protein 1B (SPRR1B), and keratin 16 (KRT16). Involvement of these pathways suggests squamous metaplasia of the epithelium. Histological analyses of epithelium in subglottic mucosal biopsies revealed squamous metaplasia in 41% of the samples from iSGS patients and in 25% from controls. Immunohistochemical evaluation of the samples presented with squamous epithelium revealed increased expression of the protein encoded by SPRR1B, hyperproliferative basal cells, shedding of apical layers, and accompanying lesions in iSGS compared to CTRL. Cultured primary subglottic epithelial cells from iSGS patients had higher proliferation rates compared to healthy donors and squamous metaplastic differentiation formed thinner epithelia with increased expression proteins encoded by INV, SPRR1B, and KRT16, suggesting intrinsic dysfunction of basal cells in iSGS. CONCLUSIONS: Abnormal squamous differentiation of epithelial cells may contribute to the pathogenesis of iSGS. Patients having metaplastic epithelial phenotype may be sensitive to drugs that reverse it to a normal phenotype.

Regenerating airway epithelium using fibrous biomimetic basement membranes.

There are reciprocal interactions between epithelial cells and underlying basement membrane. The resemblance of biomaterials to native basement membrane is thus critical for their success when used to regenerate epithelium-containing organs. Particularly, the use of nanofibers and the incorporation of basement membrane proteins may mimic both biophysical and biochemical properties of basement membrane, respectively. Herein we tested how electrospun polycaprolactone/heparin fibers with and without adsorbed laminin and collagen IV proteins affect epithelial cell functions. We found that airway epithelial cells attached, migrated, and proliferated on all scaffolds but protein-functionalized fibers promoted higher attachment, quicker migration, and increased proliferation. Fibers were then integrated on polyethylene scaffolds and cultured at an air-liquid interface. The detection of secretory and ciliated cell markers was higher in cells on polyethylene with fibers. These findings demonstrate that electrospun fibers incite beneficial epithelial cell responses and can be used in the fabrication of bioengineered functional epithelia.

Human amniotic epithelial cell transplantation improves scar remodeling in a rabbit model of acute vocal fold injury: a pilot study.

OBJECTIVE: To gain insight into the molecular mechanisms underlying the early stages of vocal fold extracellular matrix (ECM) remodeling after a mid-membranous injury resulting from the use of human amniotic epithelial cells (hAEC), as a novel regenerative medicine cell-based therapy. METHODS: Vocal folds of six female, New Zealand White rabbits were bilaterally injured. Three rabbits had immediate bilateral direct injection of 1 × 106 hAEC in 100 µl of saline solution (hAEC) and three with 100 µl of saline solution (controls, CTR). Rabbits were euthanized 6 weeks after injury. Proteomic analyses (in-gel trypsin protein digestion, LC-MS/MS, protein identification using Proteome Discoverer and the Uniprot Oryctolagus cuniculus (Rabbit) proteome) and histological analyses were performed. RESULTS: hAEC treatment significantly increased the expression of ECM proteins, elastin microfibril interface-located protein 1 (EMILIN-1) and myocilin that are primarily involved in elastogenesis of blood vessels and granulation tissue. A reactome pathway analysis showed increased activity of the anchoring fibril formation by collagen I and laminin, providing mechanical stability and activation of cell signaling pathways regulating cell function. hAEC increased the abundance of keratin 1 indicating accelerated induction of the differentiation programming of the basal epithelial cells and, thereby, improved barrier function. Lastly, upregulation of Rab GDP dissociation inhibitor indicates that hAEC activate the vesicle endocytic and exocytic pathways, supporting the exosome-mediated activation of cell-matrix and cell-to-cell interactions. CONCLUSIONS: This pilot study suggests that injection of hAEC into an injured rabbit vocal fold favorably alters ECM composition creating a microenvironment that accelerates differentiation of regenerated epithelium and promotes stabilization of new blood vessels indicative of accelerated and improved repair.

A Tissue Engineered Construct for Laryngeal Regeneration: A Proof-of-Concept Device Design Study.

OBJECTIVES/HYPOTHESIS: Develop a patient-specific tissue engineered construct for laryngeal reconstruction following a partial laryngectomy. STUDY DESIGN: Bench and animal research. METHODS: A construct made from a porous polyethylene scaffold shaped in a canine-specific configuration and seeded with autologous canine adipose-derived stem cells in fibrin glue was implanted in a canine following a partial laryngectomy. After 1 year, the construct was first evaluated in vivo with high-speed imaging and acoustic-aerodynamic measures. It was then explanted and evaluated histologically. RESULTS: The canine study at 1 year revealed the construct provided voicing (barking) with acoustic and aerodynamic measures within normal ranges. The canine was able to eat and breathe normally without long-term support. The construct was integrated with epithelialization of all areas except the medial portion of the vocal fold structure. No anti-infective agents were needed after the standard perioperative medications were completed. CONCLUSION: This study provided a successful first step toward developing a patient-specific composite construct for patients undergoing partial laryngectomies. LEVEL OF EVIDENCE: NA Laryngoscope, 132:S1-S11, 2022.

Computed Tomography Data to Generate a Reproducible, Anatomically Accurate Hemilaryngeal Model.

OBJECTIVE: The study aims to demonstrate the reproducibility and feasibility of creating a hemilaryngeal model with a medialized vocal fold (VF) using 3-dimensional (3D) modeling techniques in both healthy larynges and those affected by cancer. STUDY DESIGN: Three-dimensional modeling of human larynges. SETTING: Tertiary academic referral center and regenerative medicine laboratory. SUBJECTS AND METHODS: Computed tomography (CT) scans from 10 healthy control and 10 patients with laryngeal cancer were segmented and imported into 3D modeling software. The larynx was cut sagittally to create a hemilaryngeal model and the vocal fold medialized. Measurements were taken from the CT and 3D model data and compared. RESULTS: All control modeling data closely matched the CT data and were not statistically different from each other. There was a significant correlation between subglottic anteroposterior diameter and VF length (r2 = 0.78, P = .0008), and it may be a valuable tool to infer true VF dimension in cases where disruption has occurred. The modeling data from patients with cancer did not show statistical difference to the control data, showing that accurate modeling can also be achieved in patients with laryngeal cancer. CONCLUSION: CT scan-based 3D modeling of the larynx and VF is possible and reproducible. The results closely match those previously reported in the literature and can also be replicated in cases with laryngeal cancer. This study paves the way for future de novo fabricated laryngeal scaffolds that can be synthesized using 3D printers and tailored to meet surgical demands.

Development of xeno-free epithelial differentiation media for adherent, non-expanded adipose stromal vascular cell cultures.

INTRODUCTION: Reconstruction of respiratory epithelium is critical for the fabrication of bioengineered airway implants. Epithelial differentiation is typically achieved using bovine pituitary extract (BPE). Due to the xenogenic nature and undefined composition of BPE, an alternative for human clinical applications, devoid of BPE, must be developed. The goal of this study was to develop two different BPE-free media, with and without select pituitary hormone (PH), which could initiate epithelial differentiation for use in human implantation. METHODS: The ability of the two BPE-free media to initiate epithelial differentiation of adherent, non-expanded stromal-vascular cells grown on porcine small intestinal submucosa was compared to traditional BPE-containing media (M1). Nanostring® was used to measure differences in gene expression of stemness (MSC), basal cell (basal), and ciliated markers (muco-cil), and staining was performed support the gene data. RESULTS: Compared to baseline, both BPE-free media upregulated epithelial and stemness genes, however this was to a lower degree than BPE-containing media. In general, the expression of basal cell markers (COL17A1, DSG3, ITGA6, KRT6A, LOXL2) and secreted mucous proteins (PLUNC, MUC5B, SCGB2A1) was upregulated. The gene expression of ciliated markers C9orf24, TUBA3 and DNCL2B but not of the key transcription factor for cilagenesis FOXJ1 were upregulated, indicating that mucus-secreting cell differentiation occurs more rapidly than ciliogenesis. The ability of the adherent stromal vascular cells to upregulate gene expression of both epithelial and stemness markers suggests maintenance of the self-renewal capacity of undifferentiated and/or basal cell-like cells contributing to proliferation and ensuring a persisting source of cells for regenerative medicine applications. CONCLUSION: This study provides the initial step to defining a BPE-free epithelial differentiation medium for clinical translation. Thus, either of the proposed BPE-free medium are viable alternatives to BPE-containing medium for partial epithelial differentiation for human translational applications.

Tracheal decellularization using a combination of chemical, physical and bioreactor methods.

INTRODUCTION: The purpose of this study was to compare different decellularization protocols with the conventional detergent enzymatic method (DEM) using continuous agitation. METHODS: The first experiment compared conventional DEM with sonication and lyophilization+freeze-thaw cycles. A second experiment was carried out to compare time-adjusted DEM (2-hour instead of 4-hour incubations with 4% deoxycholate) to decellularization in a bioreactor. Cellularity was determined by DNA-quantitation, H&E-staining and immunostaining for major histocompatibility complex-1 (MHC-1). RESULTS: Compared to untreated trachea, DNA content significantly decreased after 2 cycles in all groups in the first experiment and dropped below the minimal criteria for efficient decellularization (<50 ng dsDNA/mg dry weight) after 4 cycles. However, nuclei were seen in the cartilage and MCH-1 staining was detected in some submucosal areas, indicating presence of chondrocytes and cellular residues that may render the scaffold immunogenic. In the second experiment DNA content significantly decreased after 1 cycle in both groups; however, even after 4 cycles, DNA content was above the minimal criteria for efficient decellularization. While collagen-levels remained stable, glycosaminoglycans diminished significantly after the initial cycles. CONCLUSIONS: Efficient decellularization can be achieved after only 4 cycles of DEM compared to the 17 cycles previously reported. The use of a bioreactor can preserve the integrity of the extracellular matrix.

Potential effects of aerobic exercise on the expression of perilipin 3 in the adipose tissue of women with polycystic ovary syndrome: a pilot study.

OBJECTIVE: Polycystic ovary syndrome (PCOS) is associated with reduced adipose tissue lipolysis that can be rescued by aerobic exercise. We aimed to identify differences in the gene expression of perilipins and associated targets in adipose tissue in women with PCOS before and after exercise. DESIGN AND METHODS: We conducted a cross-sectional study in eight women with PCOS and eight women matched for BMI and age with normal cycles. Women with PCOS also completed a 16-week prospective aerobic exercise-training study. Abdominal subcutaneous adipose tissue biopsies were collected, and primary adipose-derived stromal/stem cell cultures were established from women with PCOS before 16 weeks of aerobic exercise training (n=5) and controls (n=5). Gene expression was measured using real-time PCR, in vitro lipolysis was measured using radiolabeled oleate, and perilipin 3 (PLIN3) protein content was measured by western blotting analysis. RESULTS: The expression of PLIN1, PLIN3, and PLIN5, along with coatomers ARF1, ARFRP1, and ßCOP was ∼ 80% lower in women with PCOS (all P<0.05). Following exercise training, PLIN3 was the only perilipin to increase significantly (P<0.05), along with coatomers ARF1, ARFRP1, ßCOP, and SEC23A (all P<0.05). Furthermore, PLIN3 protein expression was undetectable in the cell cultures from women with PCOS vs controls. Following exercise training, in vitro adipose oleate oxidation, glycerol secretion, and PLIN3 protein expression were increased, along with reductions in triglyceride content and absence of large lipid droplet morphology. CONCLUSIONS: These findings suggest that PLIN3 and coatomer GTPases are important regulators of lipolysis and triglyceride storage in the adipose tissue of women with PCOS.

Sex dimorphism and depot differences in adipose tissue function.

Obesity, characterized by excessive adiposity, is a risk factor for many metabolic pathologies, such as type 2 diabetes mellitus (T2DM). Numerous studies have shown that adipose tissue distribution may be a greater predictor of metabolic health. Upper-body fat (visceral and subcutaneous abdominal) is commonly associated with the unfavorable complications of obesity, while lower-body fat (gluteal-femoral) may be protective. Current research investigations are focused on analyzing the metabolic properties of adipose tissue, in order to better understand the mechanisms that regulate fat distribution in both men and women. This review will highlight the adipose tissue depot- and sex-dependent differences in white adipose tissue function, including adipogenesis, adipose tissue developmental patterning, the storage and release of fatty acids, and secretory function. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease.

In vivo adipogenesis in rats measured by cell kinetics in adipocytes and plastic-adherent stroma-vascular cells in response to high-fat diet and thiazolidinedione.

Impairment of adipogenesis contributes to the development of obesity-related insulin resistance. The current in vitro approaches for its assessment represent crude estimates of the adipogenic potential because of the disruption of the in vivo microenvironment. A novel assessment of in vivo adipogenesis using the incorporation of the stable isotope deuterium ((2)H) into the DNA of isolated adipocytes and stroma-vascular fraction from adipose tissue has been developed. In the current study, we have refined this technique by purifying the adipocytes via a negative immune selection and sorting the plastic adherent stroma-vascular (aSV) subfraction (using 3 h culture) that contains mostly adipocyte progenitor cells and ∼10% of small adipocytes. Using a 3-week 8% (2)H(2)O ingestion with a high-fat diet (HFD) or HFD plus pioglitazone (HFD-P), we demonstrate that the fractions of new aSV cells (f(aSV)) and immunopurified adipocytes (f(AD)) (the ratio of their (2)H-enrichment of DNA to the maximal (2)H-enrichment of DNA of bone marrow reference cells) recapitulate the known hyperplastic mechanism of weight gain with pioglitazone treatment. We conclude that f(aSV) and f(AD) are reliable indices of in vivo adipogenesis. The proposed method represents a valuable tool for studying the effect of interventions (drugs, diets, and exercise) on in vivo adipogenesis.

Implications of 2H-labeling of DNA protocol to measure in vivo cell turnover in adipose tissue.

Adipose tissue expansion in obesity involves a series of cycles of adipocyte hyperplasia, hypertrophy and hypoplasia due to alterations in adipogenesis, adipocyte cellular metabolism and cell death, respectively. Increased frequency of these cycles may lead to deterioration of adipocyte function and viability, accelerated exhaustion of the adipocyte progenitor pool and extensive adipose tissue remodeling, all leading to impaired expandability of subcutaneous adipose tissue, ectopic lipid accumulation and insulin resistance. Understanding the mechanisms that contribute to adipocyte turnover is thus important. We have recently refined and published an existing method to assess in vivo adipogenesis using incorporation of the stable isotope deuterium into the DNA of isolated adipocytes and adipocyte progenitors from adipose tissue. In this commentary, we highlight further implications of this method to determine the rate of adipocyte hypertrophy and adipocyte death that will enhance our understanding of adipocyte cell turnover and cellular mechanisms that control regional adipose tissue growth.

Regional differences in cellular mechanisms of adipose tissue gain with overfeeding.

Body fat distribution is an important predictor of the metabolic consequences of obesity, but the cellular mechanisms regulating regional fat accumulation are unknown. We assessed the changes in adipocyte size (photomicrographs) and number in response to overfeeding in upper- and lower-body s.c. fat depots of 28 healthy, normal weight adults (15 men) age 29 ± 2 y. We analyzed how these changes relate to regional fat gain (dual energy X-ray absorptiometry and computed tomography) and baseline preadipocyte proliferation, differentiation [peroxisome proliferator-activated receptor-γ2 (PPARγ2) and CCAAT/enhancer binding protein-α (C/EBPα) mRNA]), and apoptotic response to TNF-α. Fat mass increased by 1.9 ± 0.2 kg in the upper body and 1.6 ± 0.1 kg in the lower body. Average abdominal s.c. adipocyte size increased by 0.16 ± 0.06 µg lipid per cell and correlated with relative upper-body fat gain (r = 0.74, P < 0.0001). However, lower-body fat responded to overfeeding by fat-cell hyperplasia, with adipocyte number increasing by 2.6 ± 0.9 × 10(9) cells (P < 0.01). We found no depot-differences in preadipocyte replication or apoptosis that would explain lower-body adipocyte hyperplasia and abdominal s.c. adipocyte hypertrophy. However, baseline PPARγ2 and C/EBPα mRNA were higher in abdominal than femoral s.c. preadipocytes (P < 0.005 and P < 0.03, respectively), consistent with the ability of abdominal s.c. adipocytes to achieve a larger size. Inherent differences in preadipocyte cell dynamics may contribute to the distinct responses of different fat depots to overfeeding, and fat-cell number increases in certain depots in adults after only 8 wk of increased food intake.

Sex- and depot-dependent differences in adipogenesis in normal-weight humans.

To elucidate cellular mechanisms of sex-related differences in fat distribution, we determined body fat distribution (dual-energy X-ray absorptiometry and single-slice abdominal computed tomography (CT)), adipocyte size, adipocyte number, and proportion of early-differentiated adipocytes (aP2(+)CD68(-)) in the stromovascular fraction (SVF) in the upper and lower body of normal-weight healthy men (n = 12) and premenopausal women (n = 20) (age: 18-49 years, BMI: 18-26 kg/m(2)). Women had more subcutaneous and less visceral fat than men. The proportion of early differentiated adipocytes in the subcutaneous adipose tissue SVF of women was greater than in men (P = 0.01), especially in the femoral depot, although in vitro adipogenesis, as assessed by peroxisome proliferator activated receptor-γ (PPARγ) expression, was not increased in femoral preadipocytes cultured from women compared with men. In women, differentiation of femoral preadipocytes was less than that of abdominal subcutaneous preadipocytes (P = 0.04), and femoral subcutaneous preadipocytes tended to be more resistant to tumor necrosis factor-α (TNFα)-induced apoptosis (P = 0.06). Thus, turnover and utilization of the preadipocyte pool may be reduced in lower vs. the upper-body fat in women. Collectively, these data indicate that the microenvironment, rather than differences in inherent properties of preadipocytes between genders, may explain the gynoid obesity phenotype and higher percent body fat in women compared to men.

Differential effect of weight loss on adipocyte size subfractions in patients with type 2 diabetes.

The size of adipocytes influences their function suggesting a differential responsiveness to intervention. We hypothesized that weight loss in patients with type 2 diabetes mellitus (T2DM) predominantly decreases the size of large and very-large adipocyte subfractions in parallel with beneficial changes in serum adipokines and improved insulin sensitivity. A total of 44 volunteers from the Look Action for Health in Diabetes trial, who lost weight after 1-year of intense lifestyle intervention, were included. Insulin sensitivity (hyperinsulinemic-euglycemic clamp), size of subcutaneous abdominal adipocytes (osmium fixation), and selected serum adipokines were measured. A 13% weight loss was accompanied by 46% improvement in insulin sensitivity (increased glucose disposal rate from 5.9+/-2.2 to 8.6+/-2.7 mg/min/kg fat-free mass, P<0.05) in parallel with a 36% increase in plasma adiponectin concentration (6.1+/-3.1 to 8.3+/-3.9 microg/ml, P<0.05], but no changes in the proinflammatory cytokines interleukin-6 and tumor necrosis factor-alpha. Change in adiponectin correlated with changes in glucose disposal rate (r=0.34, P<0.05). Mean adipocyte size decreased (0.84+/-0.25 to 0.64+/-0.23 microl, P<0.05), mainly due to changes in the large adipocyte subfraction (size 0.75-0.44 microl, relative number 19-26%; P<0.05). Our data suggest that change in the large adipocyte subfraction may contribute to the improvement in insulin sensitivity via an increase in serum adiponectin. Such a relationship, which does not imply cause and effect, could not be obtained by measuring only mean adipocyte size. These data provide support for the measures of adipocyte size distribution in concert with in vitro adipokine secretion and lipolysis in future studies.

Regional variation in adipogenesis and IGF regulatory proteins in the fetal baboon.

Intrauterine growth rate is associated with body distribution in adulthood suggesting differential response of fetal fat depots to nutritional modifications. We hypothesize that there is regional differences in fetal adipogenesis, in part, due to depot-specific regulation of the availability of insulin growth factors. In near-term baboon fetuses (n=3-5), the subcutaneous abdominal vs. omental preadipocytes had (1) more extensive lipid accumulation as assessed by BODIPY (lipid staining) to DAPI (nuclei) absorbance ratios (mean+/-SEM; 0.51+/-0.21, 0.35+/-0.09, p<0.05), (2) lower (p<0.05) secretion of IGF-binding protein 4 (9.6+/-1.2 vs. 17.4+/-2.8 ng/ml) and its protease pregnancy associated plasma protein A (24.6+/-1.9 vs. 39.1+/-6.3 microIU/ml), (3) lower protein expression of IGF2 "clearance" receptor in cell lysate (0.28+/-0.03 vs. 0.53+/-0.02 OD U/mm(2), p<0.05); all variables were intermediate in femoral preadipocytes. The regional variation of the adipogenesis and the IGF regulatory pathway set the stage for differential responsiveness of fat depots to external signals.

Potential role of increased matrix metalloproteinase-2 (MMP2) transcription in impaired adipogenesis in type 2 diabetes mellitus.

We measured gene expression of paracrine regulators involved in adipocyte differentiation within the stromovascular fraction of abdominal subcutaneous adipose tissue from obese individuals with (n=30) and without (n=18) type 2 diabetes mellitus (T2DM). Despite similar adiposity by design, subjects with T2DM had larger adipocytes (0.92+/-0.28 vs. 0.75+/-0.17 microl, p<0.05) than controls. Gene expression of the adipogenic marker aP2 was lower (0.35+/-0.16 vs. 0.58+/-0.27 arbitrary units, p<0.05) whereas the expression of matricellular peptidase, MMP2 was higher (1.65+/-0.17 vs. 1.27+/-0.21, p=0.02) in T2DM vs. controls. The gene expression levels between the aP2 and MMP2 were inversely correlated (r=-0.32, p=0.03). We conclude that early steps of adipogenesis may be impaired in T2DM independently of obesity due, in part, to an upregulation of the MMP2 transcription.

Subcutaneous adipocyte size and body fat distribution.

BACKGROUND: Both body fat distribution and adipocyte size are associated with metabolic abnormalities. OBJECTIVE: We defined the extent to which subcutaneous adipocyte size is related to regional fat mass and to the sizes of adipocytes in other subcutaneous depots independent of adiposity, age, and sex. DESIGN: Data collected from 188 women and 133 men who were 18-50 y old and who had a body mass index (in kg/m2) of 18 to 50 were analyzed. The mean size of isolated subcutaneous abdominal, femoral, and gluteal adipocytes was measured by direct microscopy or by automated analysis of digital images. Visceral fat area was measured with computed tomography. Dual-energy X-ray absorptiometry was used to calculate adiposity. RESULTS: Stepwise multiple regression analyses showed that abdominal adipocyte size was associated positively with visceral and subcutaneous abdominal fat areas and negatively with lower-body fat mass as a percentage of total-body fat, after control for sex and percentage body fat. Femoral adipocyte size was related to percentage body fat (P<0.0001), whereas gluteal adipocyte size was related to visceral fat area (P=0.002), which suggests that these 2 lower-body fat depots are distinct. Analyses of data from a subset of volunteers (n=99) for whom we had adipocyte size from all 3 depots showed that adipocyte size from 1 depot could be better predicted if adipocyte size from other depots were known. CONCLUSIONS: Abdominal adipocyte size is related to body fat distribution. Adipocyte size in a person seems to be globally regulated by factors independent of variations in body fat distribution.

Abundance of two human preadipocyte subtypes with distinct capacities for replication, adipogenesis, and apoptosis varies among fat depots.

Fat depots vary in function and size. The preadipocytes that fat cells develop from exhibit distinct regional characteristics that persist in culture. Human abdominal subcutaneous cultured preadipocytes undergo more extensive lipid accumulation, higher adipogenic transcription factor expression, and less TNF-alpha-induced apoptosis than omental preadipocytes. We found higher replicative potential in subcutaneous and mesenteric than in omental preadipocytes. In studies of colonies arising from single preadipocytes, two preadipocyte subtypes were found, one capable of more extensive replication, differentiation, and adipogenic transcription factor expression and less apoptosis in response to TNF-alpha than the other. The former was more abundant in subcutaneous and mesenteric than in omental preadipocyte populations, potentially contributing to regional variation in replication, differentiation, and apoptosis. Both subtypes were found in strains derived from single human preadipocytes stably expressing telomerase, confirming that both subtypes are of preadipocyte lineage. After subcloning of cells of either subtype, both subtypes were found, indicating that switching can occur between subtypes. Thus proportions of preadipocyte subtypes with distinct cell-dynamic properties vary among depots, potentially permitting tissue plasticity through subtype selection during development. Furthermore, mesenteric preadipocyte cell-dynamic characteristics are distinct from omental cells, indicating that visceral fat depots are not functionally uniform.

Measuring committed preadipocytes in human adipose tissue from severely obese patients by using adipocyte fatty acid binding protein.

To understand the significance of the reported depot differences in preadipocyte dynamics, we developed a procedure to identify committed preadipocytes in the stromovascular fraction of fresh human adipose tissue. We documented that adipocyte fatty acid binding protein (aP2) is expressed in human preadipocyte clones capable of replication, indicating that can be used as a marker of committed preadipocytes. Because aP2 expression can be induced in macrophages, stromovascular cells were also stained for the macrophage marker CD68. We found aP2+CD68- cells (designated as committed preadipocytes) that did not have lipid droplets (true preadipocytes) and that did have lipid droplets < 6.5 microm in diameter (very immature adipocytes). Adipose tissue from subcutaneous, omental, and mesenteric depots was obtained from nine patients undergoing bariatric surgery for measurement of stromovascular cell number, the number of committed preadipocytes (aP2+CD68-), aP2+ macrophages (aP2+CD68+), and aP2- macrophages (aP2-CD68+). The number of committed preadipocytes did not differ significantly between depots but varied >20-fold among individuals. Total cell number, stromovascular cell number, and the number of aP2- macrophages was less (P < 0.05) in subcutaneous than in omental fat (means +/- SE, in millions: subcutaneous, 2.3 +/- 0.3, 1.4 +/- 0.3, and 0.17 +/- 0.08; and omental, 4.8 +/- 0.7, 3.8 +/- 0.5, and 0.34 +/- 0.06); mesenteric depot was intermediate. These data indicate that the cellular composition of adipose tissue varies between depots and between individuals. The ability to quantify committed preadipocytes in fresh adipose tissue should facilitate study of adipose tissue biology.