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Keywords:

  • Adipocytes;
  • Dedifferentiation;
  • Mesenchymal stem cells;
  • Cell culture

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Mature adipocytes are generally considered terminally differentiated because they have lost their proliferative abilities. Here, we studied the gene expression and functional properties of mature adipocytes isolated from human omental and subcutaneous fat tissues. We also focused on dedifferentiated adipocytes in culture and their morphologies and functional changes with respect to mature adipocytes, stromal-vascular fraction (SVF)-derived mesenchymal stem cells (MSCs) and bone marrow (BM)-derived MSCs. Isolated mature adipocytes expressed stem cell and reprogramming genes. They replicated in culture after assuming a fibroblast-like shape and expanded similarly to SVF- and BM-derived MSCs. During the dedifferentiation process, mature adipocytes lost their lineage gene expression profile, assumed the typical mesenchymal morphology and immunophenotype, expressed stem cell genes and differentiated into multilineage cells. Moreover, during the dedifferentiation process, we showed changes in the epigenetic status of mature adipocytes, which led dedifferentiated adipocytes to display a similar DNA methylation condition to BM-derived MSCs. Like SVF- and BM-derived MSCs, dedifferentiated adipocytes were able to inhibit the proliferation of stimulated lymphocytes in coculture while mature adipocytes stimulated their growth. Furthermore, dedifferentiated adipocytes maintained the survival and complete differentiation characteristic of hematopoietic stem cells. This is the first study that in addition to characterizing isolated and dedifferentiated adipocytes also reports on the immunoregulatory and hematopoietic supporting functions of these cells. This structural and functional characterization might have clinical applications of both mature and dedifferentiated adipocytes in such fields, as regenerative medicine. STEM CELLS 2012;30:965–974


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

For many years, adipose tissue was regarded as just a heat insulator and store of excess free fatty acids that could be released when needed. Now it is considered a critical organ involved in energy balance regulation and the immune response through intricate signals [1, 2].

Adipose tissue contains adipocytes and non-adipose cells. Mature adipocytes are functionally the most important cell type in adipose tissue. They have a typical and quite specific morphology characterized by the presence of a single, large cytoplasmic lipid droplet that accounts for approximately 90% of its volume.

The process of cellular differentiation in terminally differentiated mammalian cells is thought to be irreversible, but recent data suggest that the mature adipocytes of mice, when under physiological stimuli, are able to reversibly change their phenotypes and directly transform into cells with a different morphology and physiology [3]. This process is termed transdifferentiation and implies a phenomenon of in vivo physiological and reversible reprogramming of genes in mature cells [4]. Thus, the mature adipocytes of mice have properties similar to stem cells. The isolated mature adipocytes of mice express genes described as typical for stem cells and acquire the specific differentiation and functional properties of milk-secreting epithelial cells when transplanted into the mammary glands of pregnant mice [3].

In line with this hypothesis, the aim of this study was to examine the gene expression and functional properties of healthy human mature adipocytes, which were isolated and maintained in primary culture, and dedifferentiated adipocytes. We compared these data with those from mesenchymal stem cells (MSCs) derived from human adipose tissue and bone marrow (BM), including pluripotent differentiation abilities. Furthermore, we investigated their eventual immunomodulatory and hematopoietic supporting properties and their global DNA methylation statuses.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Isolation and Culture of Adipocytes

In accordance with the guidelines of the local ethical committee (300/DG), omental and subcutaneous fat tissue (5–10 g) from patients (n = 12 + 12, 53–81 years old) were obtained at the time of abdominal surgery. The patients were not obese and undergoing surgery for early gastric or early colon-rectal cancers. Adipose tissue was promptly washed with Dulbecco's modified Eagle's medium (DMEM; Biological Industries, cat. L0064-500, Milan, Italy), and visible blood vessels were removed. The samples were minced into smaller pieces and treated with 3 mg/ml type I collagenase (Gibco, Invitrogen, Milan, Italy) at 37°C for 2 hours.

To obtain isolated mature adipocytes free of stromal-vascular elements, after collagenase digestion, the disrupted tissues were filtered through a 200-μm nylon sieve. The filtered cells were washed four times with DMEM and centrifuged at 250g for 5 minutes. Only the floating top layer was collected after each centrifugation step, which allowed for the isolation of a pure fraction of floating adipocytes and a pellet containing stromal-vascular fraction (SVF) cells. After the last centrifugation step, the fatty layer was transferred to an inverted 25-cm2 cell culture flask, completely filled with DMEM supplemented with 20% fetal bovine serum (FBS, Stem Cell Technologies, Vancouver, Canada) and seeded for ceiling culture, that is, the bottom of the flask is on top [5]. Only mature adipocytes free of any detectable contamination of stromal-vascular elements were allowed to adhere to the top layer of the flask in a 5% CO2 incubator (37°C), which was monitored daily for cell attachment. During the first 3-4 days of ceiling culture, mature adipocytes adhered loosely to the ceiling surface. From day 5 or 6, the cells started to lose their spherical shapes. After sufficient attachment of cells (usually 8-10 days), the medium was removed, replaced with fresh medium and the flask was reinverted. This protocol allowed for normal observations and the subsequent manipulation of the cultures. When the cells lost their spherical shape, they were trypsinized and subcultivated until senescence, with an initial seeding density of 2 × 103 cells per square centimeter. The cellular senescence ratio was determined by changes in cell morphology and the inability of the cells to proliferate and reach confluence in the flask. Indeed, senescent cells were larger and flatter than their actively dividing counterparts. The cells then progressively died during the senescence phase and spontaneously detached from the flasks.

After obtaining written informed consent and in accordance with the guidelines of the local ethical committee, BM was harvested from the iliac crest of six healthy donors (median age 17 years, range, 16-45 years). The data referring to bone marrow MSCs were derived from the study by Poloni et al. [6], where we used the same samples.

Omental and subcutaneous SVF-derived and BM-derived MSCs were cultured with the same medium described above and reseeded (2 × 103 cells per square centimeter) after trypsinization until the cells reached senescence.

The proliferative potential, calculated as population doublings (PDs), of the cultured cells was performed at every passage according to the equation: log2(number of harvested cells/number of seeded cells). Finite PDs were determined by the cumulative addition of the total numbers generated from each passage until the cells ceased dividing.

Immunofluorescence of Isolated Adipocytes

Confocal microscopy optical sectioning and computer-assisted image reconstruction of isolated adipocytes allowed us to exclude the presence of small undifferentiated cells attached to the surface of the analyzed adipocytes. Briefly, isolated adipocytes were fixed in 4% paraformaldehyde, incubated with rabbit anti-perilipin (kindly provided by Dr. Greenberg, Tuft University, Boston, ME, 1:50 in phosphate-buffered saline [PBS]) and stained with fluorescein isothiocyanate (FITC)-linked secondary antibodies (Jackson ImmunoResearch, West Grove, PA, 1:100 in PBS). Images were taken in the green channel. TOTO-3 iodide (Molecular Probes, Invitrogen, 1:5,000 in PBS) was used as the nuclear counterstain and visualized in the blue channel. We analyzed at least 100 adipocytes from each suspension. Images were sequentially obtained from two channels using a pinhole of 1.1200. The brightness and contrast of the final images were adjusted using Photoshop six software (Adobe Systems; Mountain View, CA).

Quantitative Cytokine Assay

When cells reached confluence, the culture supernatants of dedifferentiated adipocytes at different passages were collected and frozen at −20°C. Multiplex human cytokine, chemokine and growth factor detection (BioPlex, BioRad, Segrate, Italy) were used to measure the production of interleukin (IL)-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, basic fibroblast growth factor (FGF-β), eotaxin, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), interferon-γ-induced protein (IP-10), macrophage chemotactic protein (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1β, platelet-derived growth factor-bb, CCL5 (RANTES), tumor necrosis factor-α (TNF-α), and vascular endothelium growth factor (VEGF) in the culture supernatants.

Electron Microscopy

After ceiling culture, dedifferentiated adipocytes as well as the SVF-derived MSCs obtained from the sampled fat tissues [7, 8] were fixed in 2% glutaraldehyde + 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for at least 4 hours, postfixed in a solution of 1% osmium tetroxide and 1% potassium hexacyanoferrate (II), dehydrated in acetone and finally epoxy-resin embedded. Semithin sections (2 μm) were stained with toluidine blue. Thin sections obtained with an MT-X ultratome (RCM, Tucson, AZ) were mounted on copper grids, stained with lead citrate and examined with a CM10 transmission electron microscope (Philips, Eindhoven, The Netherlands).

Immunophenotype Analysis

After isolation, seven samples of mature adipocytes were analyzed by flow cytometry. Cells were stained with FITC-, phycoerythrin (PE-) or peridin chlorophyll protein (PerCP-) conjugated antibodies against CD34 (hematopoietic progenitor cell antigen, BD Biosciences, Franklin Lakes, NJ), CD117 (c-kit, Miltenyi, Biotech, Cologne, Germany), CD271 (NGFR, Miltenyi), CD133 (hematopoietic stem cell antigen, Miltenyi), CD45 (leukocyte common antigen, BD Biosciences), and CD90 (Thy-1, BD Pharmingen, San Diego, CA).

Seven samples of dedifferentiated adipocytes were characterized by flow cytometry after their fourth passages. Cells were trypsinized and stained with FITC-, PE-, or PerCP- conjugated antibodies against CD34, CD117, CD271, CD133, CD45, CD90, CD105 (endoglin), CD73 (ecto-5′-nucleotidase), CD44 (hyaluronate receptor), CD29 (integrin β1), CD14 (monocyte/macrophage marker, BD Biosciences), and HLA-DR (human leukocyte antigen-DR, BD Biosciences).

FITC, PE (Dako), and PerCP (Becton Dickinson Pharmingen) negative isotypes were used as control antibodies. Cells were incubated with primary antibodies at 4°C for 30 minutes. Thereafter, cell fluorescence was evaluated by flow cytometry using a FACSCalibur instrument (Becton Dickinson). The data were analyzed using CellQuest Software.

Adipogenic, Osteogenic, Chondrogenic, and Neurogenic Differentiation

Omental and subcutaneous samples (n = 4) of dedifferentiated adipocytes were cultured in differentiation medium after four passages. Cells were cultured in adipogenic (NH AdipoDiff; Miltenyi Biotech), osteogenic (NH OsteoDiff; Miltenyi Biotech), and chondrogenic medium (NH ChondroDiff; Miltenyi Biotech). Cells were diluted to final concentrations of 7.5 × 103, 4.5 × 103, and 3.75 × 105 cells per square centimeter in differentiation medium and cultured for 21, 10, and 24 days, respectively. Samples were performed in duplicate, and cells maintained in regular medium were used as a negative control.

Intracellular lipid droplets indicated adipogenic lineage differentiation. The differentiation potential in the osteogenic lineage was evaluated by calcium accumulation, as assessed by Alizarin Red (Sigma-Aldrich, St. Louis, MO). Chondrogenic differentiation was evaluated using a kit (DakoCytomation LSAB System-AP; Dako, Glostrup, Denmark) for the detection of aggrecan. The differentiation potential of dedifferentiated adipocytes (n = 4) into a neurogenic lineage was studied after four passages. To induce differentiation, 5 × 104 cells per square centimeter were cultured for 7-14 days in the presence of complete neural proliferation medium (NeuroCult NS-A basal medium + NeuroCult NS-A proliferation supplements, 1:10; Stem Cell Technologies) supplemented with 10 μg/ml human epidermal growth factor, 10 μg/ml human fibroblast growth factor β, and 2 mg/ml heparin. When neurospheres were formed in suspension, which was usually after 7-14 days of culturing, they were harvested and subcultivated in the same medium. The neurospheres were used to evaluate nestin expression levels using real-time polymerase chain reaction (PCR).

Reverse-Transcription PCR

Total RNA was extracted using the RNeasy Plus Micro Kit (Qiagen, Milan, Italy) according to the manufacturer's instructions. The purity of the RNA was confirmed by determining the 260/280 nm absorbance ratio (>1.8). For each sample, 1 μl of total RNA was reverse transcribed in a 20 μl reaction containing 5× reaction buffer (Invitrogen, Life Technologies, Monza, Italy), 100 mM nucleotide triphoshates (dNTPs) (Biotech, Milan, Italy), 50 mM MgCl2 (Promega, Milan, Italy), 3 μg/μl random hexamers (Invitrogen), 100 mM dithiothreitol (DTT) (Invitrogen), 40 U/μl RNase inhibitor (Takara, Shiga, Japan), and 200 U/μl moloney murine leukemia virus reverse transcriptase (MMLV) reverse transcriptase (Invitrogen). Reactions proceeded for 10 minutes at 70°C, 10 minutes at 20°C, 45 minutes at 42°C, and 3 minutes at 99°C. For each PCR reaction, 1.5 μl cDNA was amplified in a 25-μl reaction using 10× buffer (Euroclone, Milan, Italy), 50 mM MgCl2 (Euroclone), 10 mM dNTPs (Biotech), 10 μM sense and antisense gene-specific-primers and 5 U/μl Taq (Euroclone). Primers for Nanog (NM_024865.2; 213 bp), T-box 1 (Tbx1) (NM_005992.1; 213 bp), sex determining region Y-box 17 (Sox17) (NM_022454.3; 154 bp), Gata4 (NM_002052.3; 194 bp), octamer-binding transcription factor 4 (Oct4) (NM_002701.4; 125 bp), oncogene myc (c-myc) (NM_002467.4; 248 bp), kruppel-like factor four (Klf4) (NM_004235.4; 169 bp), SRY (sex determining region Y)-box 2 (Sox2) (NM_003106.3; 191 bp), hematopoietic progenitor cell antigen (CD34) (NM_001025109.1; 165 bp), c-kit (CD117) (NM_000222.2; 179 bp), nerve growth factor receptor (CD271) (NM_002507.3; 186 bp), prominin 1 (CD133) (NM_006017.2; 195 bp), leukocyte common antigen (CD45) (NM_002838.3, 153 bp), Thy-1 cell-surface antigen (THY1, CD90) (NM_006288.3, 167 bp), adiponectin (Adipoq) (NM_004797.2; 173 bp), fatty acid binding protein four (aP2) (NM_001442.2, 133 bp), pre-adipocyte factor 1 (Pref1) osteopontin (OPN) (NM_000582.2; 162 bp), osteocalcin (OC) (NM_199173; 355 bp), sex determining region Y-box 9 (Sox-9) (NM_000346; 169 bp), collagen II (COL2A1) (NM_001844; 213 bp) (NM_003836.5, 147 bp), and nestin (NES) (NM_006617.1, 179 bp) were designed using Primer3 software (http://frodo.wi. mit.edu/primer3/) and primer pairs designed to span at least one exon-intron junction to exclude any possibility of genomic DNA amplification. PCR conditions consisted of an initial denaturation at 94°C for 4 minutes followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing for 45 seconds, extension at 72°C for 45 seconds and a final extension step at 72°C for 7 minutes. The annealing temperature for all primers was 60°C. PCR products were separated by electrophoresis in 2% agarose gel, then stained with ethidium bromide and visualized using an UV illuminator.

Real-Time PCR for Quantification of Adiponectin, aP2, Pref1, and Nestin Transcripts

Real-time PCR assays with four samples each of mature adipocytes, dedifferentiated adipocytes, SVF-derived MSCs, and BM-derived MSCs were performed to quantify the Adipoq, aP2, and Pref1 transcripts. NES measured the capacity of dedifferentiated adipocytes to differentiate into a neurogenic lineage.

The SsoFast EvaGreen supermix and primers listed above were optimized for the Bio-Rad iCycler optical module system in a 20-μl mixture, containing 10 μl of supermix, 10 μM of each primer, and 2 μl of cDNA template. After 30-second at 95°C, 40 cycles of denaturation, 5-second at 95°C and annealing/extension for 10-second at 60°C were run. A melt curve was performed using one cycle at 65°C-95°C with a 0.5°C increase 10-second per step. Samples were performed in duplicate. The transcript levels of Adipoq, aP2, and Pref1 were normalized for the expression of IPO8 (NM_006390.3, 185 bp) and FBXL10 (NM_032590.4, 184 bp), constitutive genes and an endogenous calibrator (BM-MSCs) following the 2−ΔΔCt method [9]. The transcription level of nestin was normalized for the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (NM_002046.3) and β-actin (NM_001101.3). These internal control genes were chosen as references genes without a validation of their reliability specific for these set of experiments. This is a methodological limitation.

Mature and Dedifferentiated Adipocytes and Lymphocyte Cocultures

In accordance with the guidelines of the local ethical committee (300/DG), peripheral blood mononuclear cells (PB-MNCs) were obtained from healthy donors at the Transfusion Medicine Unit of our hospital and plated at a concentration of 105 cells/100 μl. PB-MNCs were stimulated with 10 μg/ml phytohemagglutinin (Sigma-Aldrich). Mature and dedifferentiated adipocytes attached to thin glass slides were cocultured with lymphocytes at a ratio of 1:10 in RPMI 1640 (Biological Industries) with 10% heat-inactivated FBS at different time points, which were days 8, 16, 24, and after trypsinization. Cultures were incubated at 37°C in 5% CO2 for 4 days and then stained overnight with bromodeoxyuridine (BrdU) (Roche, Mannheim, Germany). Absorbances were measured using a 1420 Multilabel Counter Victor3 luminometer (Perkin Elmer, Germany). The stimulatory or inhibitory effect of adipocytes on the proliferation of lymphocytes was calculated using the following formula: % stimulation or inhibition of lymphocyte proliferation (SP) or (IP) = (proliferation of lymphocytes with mitogens in the presence of adipocytes/proliferation of lymphocytes with mitogens) × 100.

Methylated DNA Quantification Assay

This assay was performed for detecting global DNA methylation status using DNA isolated from selected mature adipocytes, dedifferentiated adipocytes, and BM-derived MSCs. The amount of DNA for each assay was 50-200 ng (optimal quantification 100 ng). DNA methylation occurs due to the covalent addition of a methyl group at the 5-carbon of the cytosine ring by DNA methyltransferases, which results in 5-methylcytosine (5-mC). Thus, the quantification of 5-mC content was assessed using a Methylflash Methylated DNA Quantification kit (Epigentek, Farmingdale, NJ) according to the manufacturer's procedures. Both negative and positive DNA controls were provided in the kit. In this assay, DNA was bound to strip wells that were specifically treated to have a high DNA affinity. The methylated fraction of DNA was detected using capture and detection antibodies and then quantified colorimetrically by reading the absorbance in a microplate 1420 Multilabel Counter Victor3 spectrophotometer (Perkin Elmer). The amount of methylated DNA was proportional to the optical density (OD) intensity measured. The % 5-mC was calculated using the following formula: ([sample OD-negative control OD] ÷ S/[positive control OD-negative control OD] × 2 ÷ P) × 100, where S was the amount of input sample DNA in nanograms and P was the amount of input positive control DNA in nanograms.

Long-Term Culture-Initiating Cells

Long-term culture-initiating cell (LTC-IC) was performed on dedifferentiated adipocytes, SVF-derived MSCs, and a murine M2 B10 feeder layer (Vancouver, British Columbia, Canada), which was used as a positive control, in 24-well plates. In accordance with the guidelines of the local ethical committee (300/DG), hematopoietic CD133+ stem cells were isolated using leukapheresis from patients undergoing autologous transplantations by immunomagnetic separation (CD133+ Progenitor Cell Isolation kit, Miltenyi Biotech). Irradiated dedifferentiated adipocytes, SVF-derived MSCs, and murine cells (80 Gy) were seeded at 5 × 104 per well in a final volume of 1 ml of MyeloCult H5100 (Stem Cell Technologies) supplemented with 1% hydrocortisone. CD133+ cells were seeded at concentrations of 1000, 500, 250, and 125 cells per well. Cultures were performed in quadruplicate and incubated at 33°C in a fully humidified atmosphere with 5% CO2. Cultures were fed every week by a half-medium change. The total progenitor cell content of each well was assessed by trypsinizing wells after 8 weeks of culturing and plating cells in methylcellulose in 35-mm tissue culture dishes BD (Falcon, Milan, Italy). Assays for myeloid and erythroid progenitors (colony-forming unit-granulocyte-macrophage [CFU-GM]; burst-forming unit-erythroid, BFU-E) were performed in a single-layer methylcellulose using HSC-CFU Lite (Miltenyi Biotech) containing stem cell factor (SCF), IL-3, GM-CSF, and erythropoietin. The cultures were incubated at 37°C in a fully humidified atmosphere with 5% CO2 for 14 days.

Statistical Analysis

The data are presented as means ± standard deviations. We analyzed normally distributed data using Student's t test double tailed. A nonparametric test, Wilcoxon Rank test, was used for abnormally distributed results. Differences were considered statistically significant at p < .05.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Human Mature Adipocytes Isolated from Visceral and Subcutaneous Adipose Tissues Express Stem Cell Genes and Reprogramming Genes

In a previous work [3], we demonstrated the expression of stem cell genes and reprogramming genes in isolated mature murine adipocytes. In this study, we investigated whether mature human adipocytes (from subcutaneous and visceral fat) contain transcripts for embryonic stem cell genes that are required for self-renewal and pluripotency, which include Nanog, Sox17, Gata4, Tbx1 [10], and for genes required for the cell reprogramming process, which include Oct4, Klf4, c-myc, and Sox2 [11]. Furthermore, because hematopoietic stem cell transcripts were found in isolated mature murine adipocytes, we also studied the expression of CD34, CD117, CD271, CD133, CD45, and CD90.

Isolated adipocytes floated to the top of the medium in culture flasks. Preliminary studies with confocal microscopy excluded any contamination by other cell types contained in the whole tissue. In fact, the adipose floating cell fraction did not contain any small cells among the unilocular large mature adipocytes. Next, we reconstructed the entire surface of 100 random floating adipocytes from each sample. All examined cells were large, perilipin-immunoreactive, unilocular adipocytes with a single flattened nucleus, and no other nuclei were observed adhering to the surface of the fat cells (Fig. 1). Therefore, we could conclude that 100% of the floating cells were mature adipocytes.

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Figure 1. Confocal microscopy analysis. Immunostaining in the floating adipocyte fraction for perilipin was green, and the nuclei, counterstained with TOTO-3, were blue. Scale bar = 80 μm.

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The data showed that Nanog, Tbx1, Sox17, and Gata4 were expressed in isolated mature adipocytes. Genes considered important for reprogramming the genome of mature cells (Oct4, c-myc, Klf4, and Sox2) as well as genes considered typical for hematopoietic stem cells (CD34, CD117, CD271, CD133, and CD90) were also expressed by the isolated mature adipocytes. There was no CD45 expression (Fig. 2).

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Figure 2. Molecular analysis for stem cell markers. The expression of embryonic stem cell genes (A), cell-reprogramming genes (B), and hematopoietic stem cells genes (C) was performed in mature adipocytes, dedifferentiated adipocytes after one passage of culturing, SVF-derived MSCs and BM-derived MSCs after four passages by qualitative analysis, as described in Materials and Methods. Abbreviations: BM, bone marrow; MSC, mesenchymal stem cell; and SVF, stromal-vascular fraction.

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Moreover, we expanded our study to hematopoietic and mesenchymal cell-surface antigens using the cytofluorometry of mature adipocytes. The cells tested were positive for CD34 (16% ± 0.5%), CD117 (24% ± 0.7%), CD271 (70% ± 0.3%), CD133 (15% ± 0.3%), and CD90 (45% ± 1%) but negative for CD45 (Supplementary Information 1).

Dedifferentiated Adipocytes in Ceiling Cultures Display Functional Properties Similar to Those of SVF- and BM-Derived MSCs

Ceiling cultures were performed using mature adipocytes isolated from patients with early gastric and colon-rectal cancer that were localized and did not enlarge to omental and subcutaneous fat tissues. Therefore, the analyzed tissues were healthy, and significant differences in the data generated from the two sample cohorts were not observed. In the few cases with no malignant conditions, the data were not different.

From day 5 or 6, the cytoplasm of the ceiling culture began to spread. The adipocytes then changed to a fibroblast-like morphology. Upon culturing, they lost a considerable amount of lipid, the nuclei became more centralized, and the cells became elongated in shape. When the culture medium was changed and the flasks were inverted on day 8-10 of the ceiling culture (Fig. 3), the cells entered a proliferative log phase. While the mature omental and subcutaneous adipocytes did not initially replicate (days 3-4), after the assumption of a more elongated shape, the omental and subcutaneous adipocytes averaged 18 ± 5 PDs in 90 ± 24 days during six passages of culturing and 14.5 ± 7.3 PDs in 88 ± 6 days during five passages of culturing, respectively, until reaching cellular senescence. Thus, the proliferative capacities of the omental- and subcutaneous-derived dedifferentiated adipocytes were similar.

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Figure 3. Dedifferentiation process of mature adipocytes. During culturing, the cells attached to the upper surface of the flasks, followed by conversion to fibroblast-like dedifferentiated adipocytes, reached a morphology similar to BM-derived MSCs. (A): Morphological changes at different time points from mature adipocytes to dedifferentiated adipocytes. (B): Mature adipocytes loose their lipid droplets. Cells were stained by toluidine blue. Scale bar = 4 μm (B), 80 μm (A1), 40 μm (A2), 80 μm (A3), 80 μm (A4), 120 μm (A5), and 150 μm (A6).

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Moreover, to preclude any biochemical changes between the dedifferentiated adipocytes at different passage numbers, we expanded our analysis to biochemical comparisons and studied quantitative cytokine secretions. The data showed that there were no significant differences between the cells when tested at different time points (Supplementary Information 2).

There were also no significant differences in the proliferative potentials between the omental dedifferentiated adipocytes and the omental SVF-derived MSCs after the sixth culture passage (19.6 ± 3.1 PDs in 79 ± 17 days), or the subcutaneous dedifferentiated adipocytes and the subcutaneous SVF-derived MSCs after the fifth culture passage (17.6 ± 5.9 PDs in 60 ± 10 days).

Moreover, there was no significant difference between the dedifferentiated adipocytes and the SVF-derived MSCs from the omental and subcutaneous samples with respect to the BM-MSCs after six passages (21.5 ± 2.9 PDs in 62 ± 11 days) [6].

Dedifferentiated Adipocytes Display the Structural Characteristics of SVF-Derived MSCs

Electron microscopy of dedifferentiated adipocytes is shown in Figure 4A and 4B. The images show most organelles described during the early stages of developing SVF-derived MSCs in primary cultures [7, 8], that is, well-developed Golgi complexes, short strands of rough endoplasmic reticulum, small lipid droplets, small mitochondria, lysosomes, and small granules of glycogen. Nuclei were fusiform with smooth edges. Thus, the electron microscopy (EM) features of dedifferentiated adipocytes were very similar to developing SVF-derived MSCs (Fig. 4C).

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Figure 4. Electron microscopy analysis. (A): Dedifferentiated adipocytes displayed structural organelle similarities (cytoplasm enlarged in B) to SVF-derived MSCs [7, 8] (C). Abbreviations: m, mitochondria; RER, rough endoplasmic reticulum.

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Adipocytes Lose Their Lineage Gene Expression Profiles During the Dedifferentiation Process

To examine changes in the gene expression profile during dedifferentiation in culture, real-time PCR was used to analyze the expression of some mature adipose-specific markers such as Adipoq, aP2, and Pref1 in mature adipocytes, dedifferentiated adipocytes, SVF-derived MSCs, and BM-derived MSCs. Data from dedifferentiated adipocytes, SVF-derived MSCs, and BM-derived MSCs displayed a significant decrease of Adipoq, aP2, and Pref1 expression levels (p < .05) compared with mature adipocytes (Fig. 5).

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Figure 5. Molecular analysis for adipocyte markers. Expression of Adipoq, aP2, and Pref1 was performed by quantitative analysis in mature adipocytes, dedifferentiated adipocytes after one passage of culturing, SVF-derived MSCs and BM-derived MSCs after four passages. Data were analyzed using the comparative 2−ΔΔCt method as described in Materials and Methods. Samples were run in duplicate, and the values are the mean ± SD. *, p < .05. Abbreviations: BM, bone marrow; MSC, mesenchymal stem cell; and SVF, stromal-vascular fraction.

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Dedifferentiated Adipocytes Express Stem Cells Antigens

To identify protein expression at the surface of cells, dedifferentiated adipocytes after four passages were compared to BM-derived MSCs [12] and SVF-derived MSCs [13] using a flow cytometric technique. Dedifferentiated adipocytes were uniformly positive (>80%) for CD90, CD105, CD73, CD44, and CD29, which are molecules typically expressed by BM-derived MSCs, and negative (<10%) for CD34, CD117, CD133, CD271, CD45, HLA-DR, and CD14. This profile was consistent with previous findings for BM-derived MSCs [12].

Nanog, Tbx1, Sox17, and Gata4 were expressed in dedifferentiated adipocytes and in SVF-derived MSCs. In a previous work using the same culture conditions, we showed that these genes were also expressed in adult and fetal MSCs derived from human bone marrow, chorionic villi, and amniotic fluid [14].

Moreover, dedifferentiated adipocytes, SVF-derived MSCs, and BM-derived MSCs expressed cell reprogramming genes (Oct4, c-myc, Klf4, and Sox2) and hematopoietic stem cell genes (CD34, CD117, CD271, and CD90) but not CD45. The CD133 gene was expressed by SVF-derived MSCs but not by dedifferentiated adipocytes or BM-derived MSCs (Fig. 2).

Dedifferentiated Adipocytes Are Able to Differentiate into Adipogenic, Osteogenic, and Chondrogenic Lineages

After adipogenic induction, dedifferentiated adipocytes restarted to accumulate lipid vacuoles within their cytoplasm, which resulted in positive oil red O staining and a positive reverse transcriptase-PCR (RT-PCR) for Adipoq expression with a higher expression level by real-time PCR with respect to dedifferentiated adipocytes not subjected to differentiation (p < .05; data not shown).

Osteogenic differentiation was performed, which resulted in mineralized matrix aggregates stained by Alizarin Red and positive expressions for OPN and OC by RT-PCR (data not shown).

Chondrogenic differentiation was performed after chondrogenic induction in culture, which resulted in positive staining for aggrecan and the positive expression of Sox9 and COL2A1 (data not shown, Fig. 6).

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Figure 6. The capacity of dedifferentiated adipocytes to transform into different cytotypes. (A): Adipogenic differentiation staining with oil red O. (B): Osteogenic differentiation staining with Alizarin Red. (C): Chondrogenic differentiation staining with aggrecan. Scale bar = 250 μm (C) and 50 μm (A and B).

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Dedifferentiated Adipocytes Are Able to Differentiate into a Neurogenic Lineage

Some neurogenic differentiation potentials have been shown using adipose-derived mesenchymal stem cells [15]. Thus, we studied the differentiation potential of dedifferentiated adipocytes into neurogenic lineages. The tested cells displayed the capability of forming neurosphere-like structures [16] after 10-14 days of culturing in a specific medium. Real-time PCR analyses of neurosphere structures showed the significantly increased expression of nestin, a marker of neuronal progenitors, with respect to dedifferentiated adipocytes not subjected to neurogenic differentiation (p < .05). These results indicated that dedifferentiated adipocytes could undergo some specific differentiations, including neurogenic differentiation (Fig. 7).

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Figure 7. Neurogenic differentiation capacity. (A): Neurosphere structures derived from dedifferentiated adipocytes after four passages of culturing. Scale bar = 80 μm (A1) and 50 μm (A2). (B): Nestin expression was performed by quantitative analysis. Data were analyzed using the comparative 2−ΔΔCt method as described in Materials and Methods. Samples were run in duplicate, and the values are the mean ± SD. *, p < .05. Abbreviations: BM, bone marrow; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; and MSC, mesenchymal stem cell.

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Dedifferentiated Adipocytes Display the Same DNA Methylation Status As Bone Marrow-Derived MSCs

Mature adipocytes (n = 5) with a single large lipid droplet within their cytoplasm, which was isolated from the floating cell fraction, displayed 0.4 ± 0.1% methylated DNA, dedifferentiated adipocytes (n = 5) 0.015 ± 0.007%, SVF-derived MSCs (n = 5) 0.022 ± 0.005%, and BM-derived MSCs (n = 5) 0.013 ± 0.008%. Thus, there was a statistically significant difference between mature adipocytes and dedifferentiated adipocytes (p < .001) while there was no difference between dedifferentiated adipocytes, SVF-derived MSCs, and BM-derived MSCs (Supplementary Information 3).

Adipocytes Modulate Allogeneic Lymphocyte Proliferation in Direct Coculture

We analyzed the immunoregulatory capacity of mature adipocyte samples (n = 5) during the dedifferentiation process and studied their behavior in cocultures with allogeneic lymphocytes. The morphological changes of mature adipocytes observed during ceiling culturing were associated with functional changes. Indeed, dedifferentiated adipocytes were able to inhibit the proliferation of stimulated lymphocytes in coculture while mature non-dedifferentiated adipocytes stimulated their growth. The data showed that at day 8 of ceiling culturing, when most cells were mature adipocytes with a single large lipid droplet, four of the five samples induced significant allogeneic lymphocyte proliferation (SP = 33.5 ± 3%). At day 16, three of the five samples induced significant low inhibitory effects (IP = 15 ± 4%). However, at day 24 and after trypsinization, when all the cells were fibroblast-shaped, the inhibitory effect increased significantly for all samples (IP = 42 ± 5% and IP = 90 ± 2%, respectively; Supplementary Information 4).

Dedifferentiated Adipocytes Support the Survival and Complete In Vitro Differentiation of Hematopoietic Progenitors

We compared the potential of dedifferentiated adipocytes (n = 5) and SVF-derived MSCs (n = 5) to support hematopoiesis in LTC-IC, which maintained the survival and complete differentiation of hematopoietic stem cells. In the methylcellulose assay, clonogenic cells were scored at day 14 as CFU-GM or BFU-E. The total number of CFU per 1,000 initially plated CD133+ cells was 118 ± 16 in the presence of dedifferentiated adipocytes, 96 ± 12 in presence of SVF-derived MSCs, and 168 ± 26 in presence of the murine positive control M2 B10. In negative control cultures (dedifferentiated adipocytes and SVF-derived MSCs alone), hematopoietic colonies were never observed. These results demonstrated that dedifferentiated adipocytes supported the complete in vitro differentiation of hematopoietic progenitors without a significant difference in the well-known hematopoietic-supporting capacity of SVF-derived MSCs ([17], Supplementary Information 5).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Historically, adipose tissue has been thought to play a passive metabolic role, acting solely as an energy storage reservoir [1]. This view has now changed, and adipose tissue is considered an important endocrine organ that provides plastic properties [18, 19]. Adipose tissue has also recently been reported as an important reservoir of stem cells with possible practical uses in medicine [20].

The stem cell properties of SVF-derived MSCs and their potential role in regenerative medicine have gained the most attention [21]; however, many studies have actually documented their osteogenic, chondrogenic, adipogenic, and angiogenic [13, 22] differentiation properties.

Our group has also shown that adult adipocytes in vivo and under physiologic stimuli can reversibly change their phenotype and transform into new cells with different morphologies and physiologies [3, 23]. This result implies that mature adipocytes can reprogram their genomes. In line with these data, we have also shown that isolated mature murine adipocytes express stem cell genes and reprogramming genes and, when injected into the mammary glands of hosts during pregnancy and lactation, can undergo a process of differentiation (transdifferentiation) into milk-secreting epithelial glandular cells [3]. Thus, a potential role for the use of mature adipocytes as stem cells is evident from our studies and also supported, as a hypothesis, by other authors [24].

When maintained in culture, mature adipocytes undergo a process of dedifferentiation [13]. These cells can be converted into fully differentiated osteoblasts in vitro and in vivo using all-trans retinoic acid [25], into adipocytes both in vivo and in vitro [26], and into chondrocytes and skeletal myocytes in vitro [27] under the appropriate culture conditions. Dedifferentiated adipocytes also have the potential to rapidly acquire the endothelial phenotype in vitro and to promote neovascularization in ischemic tissue and vessel-like structure formation [22]. These differentiation/transdifferentiation processes may represent the manifestation of morphological, molecular, and functional changes of mature adipocytes when exposed to specific microenvironments.

Most of these data were obtained studying murine adipocytes. Here, we present results from mature adipocytes isolated from the visceral and subcutaneous adipose tissues of adult patients. Moreover, as a novelty, we report on the functional status similarities between dedifferentiated adipocytes, SVF-derived MSCs, and BM-derived MSCs and show the same immunoregulatory and hematopoiesis-supporting capacities between these tested cells. We showed that isolated mature adipocytes expressed stem cell genes as well as reprogramming genes. All stemness markers studied at the molecular level were also expressed as surface antigens, and typical mesenchymal stem cell markers were highly preserved at the molecular and antigenic levels even after the dedifferentiation process. In contrast, CD45, a hematopoietic marker, was not expressed by mature adipocytes, while CD34 and CD133 were lost as antigens during the dedifferentiation process.

Thus, the plastic properties of mature murine adipocytes might also be present in human adipocytes. In line with this hypothesis, dedifferentiated adipocytes lost mature adipocyte markers, which were expressed as typical MSC surface antigens, acquired a high proliferative potential, which led to the capacity to replicate in culture at the same rate as BM- and SVF-derived MSCs, and had the ability to differentiate into multiple cell lineages [28]. Moreover, our results indicate the capacity of the dedifferentiated adipocytes to differentiate into neurosphere-like structures. These findings suggest that the dedifferentiated adipocytes are a homogeneous population expressing the same markers as BM-MSCs and SVF-derived MSCs. This result might be interpreted as a return back to a noncommitted status for dedifferentiated adipocytes, which was favored by the culture conditions. All together these data suggest that dedifferentiated adipocytes have the molecular signature of a reprogrammed cell with features similar to stem cells. In line with our results, Ono et al. [29] have shown that mature porcine adipocytes downregulated many genes that play a major role in lipid metabolism and upregulated genes involved in cell proliferation, altered cell morphology and regulation of differentiation. These results suggest that mature porcine adipocytes re-entered the cell cycle, gained a fibroblast-like appearance and had a multipotent capacity for lineage differentiation, which is in line with our results obtained with human visceral and subcutaneous adipocytes.

The methylation status of cells is the most common epigenetic modification of the genome in mammalian cells [30]. We found a statistically significant difference between the methylation statuses of mature adipocytes and dedifferentiated adipocytes, while there was no difference between dedifferentiated adipocytes and BM-derived MSCs. Therefore, these data suggest that during the dedifferentiation process a gene reprogramming event takes place, which leads to changes in cellular epigenetic status. By this process, dedifferentiated adipocytes achieve the DNA methylation status and functional properties of BM-MSCs.

In this study, we have shown results for functional properties previously only studied in SVF- and BM-derived MSCs [31, 32]. These characteristics are typical of MSCs, and, thus, we concentrated on these cellular functional states to emphasize the similarities observed between dedifferentiated adipocytes, SVF-, and BM-derived MSCs. Therefore, in addition to a detailed structural analysis of the studied cells, we also compared dedifferentiated adipocytes for some important functional properties.

Dedifferentiated adipocytes were able to inhibit the proliferation of stimulated lymphocytes in direct coculture, while mature fat cells stimulated their growth. These features may be associated with the ability of adipose tissue to promote inflammation via cytokine production and with the immunoregulatory capacity of BM-derived MSCs [33, 34]. In a previous work, we demonstrated the immunomodulatory activity of MSCs isolated from different sources (amniotic fluid, chorionic villi, and BM) not only versus stimulated allogeneic lymphocytes but also versus immunoselected lymphocyte populations [14, 35]. Furthermore, the immunoregulatory properties of SVF-derived MSCs have been shown by many studies [36].

Moreover, we established that dedifferentiated adipocytes, such as SVF- and BM-derived MSCs, were able to maintain the survival and self-renewal of hematopoietic stem cells and to support the complete differentiation of hematopoietic progenitors. Ookura et al. [37] compared the hematopoietic-supporting capacity of adipocytes differentiated in culture and SVF-derived MSCs and their progenitors [17]. They cocultured umbilical cord blood CD34+CD38 cells on MSCs or adipocytes and found that the hematopoietic-supporting capacity of MSCs decreased with adipocyte differentiation. However, CD34+CD38 cells cocultured with adipocytes preserved their ability to engraft in NOD/SCID mice, suggesting that adipocytes maintain the ability to support transplantable SCID-repopulating cells.

Finally, despite the functional differences between visceral and subcutaneous fat revealed in vivo [38], we have not found relevant differences in isolated adipocytes or dedifferentiated adipocytes in culture, which were derived from two anatomical sites in this study. This further suggests that the reprogramming of genes in isolated as well as cultivated adipocytes is in line with the obvious plastic properties of these cells.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

In summary, this study focused on stem cell features and the properties of isolated mature human adipocytes and reports on some of the functional changes potentially involved in the dedifferentiation process. We can speculate that during this process, gene reprogramming events take place, which lead to changes in the epigenetic status of cells and allow them to acquire morphological and functional stem cell properties. Specific immunomodulatory properties and the hematopoietic regulation of dedifferentiated adipocytes have also been shown to support the idea that a functional relationship among a bone marrow hematopoietic system, fat-lymphatic system (mesentery and omentum) and adipocytes accounts for their well-known anatomical relationship.

These experimental procedures will be of use not only for a better understanding of the mechanism of dedifferentiation, controlling and possibly altering the plasticity of the differentiated cells, but also for applications in regenerative medicine and cell-based therapies. Therefore, further studies are needed to demonstrate the molecular mechanisms that establish and maintain self-renewal, pluripotency, and other stem cell features during the isolation and dedifferentiation process of adipocytes.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

This work was supported by grants from Associazione Italiana contro le leucemie, linfomi e mieloma (AIL), sezione di Ancona-ONLUS.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
STEM_1067_sm_SuppFig1.tif857KSupplementary 1. FACS analysis of surface antigen markers on mature adipocytes. The open histograms indicate negative controls.
STEM_1067_sm_SuppFig2.tif573KSupplementary 2. Levels of factors secreted from omental (A) and subcutaneous (B) dedifferentiated adipocytes at three different time points. There were no significant differences between omental and subcutaneous cells. Results from the averages of three samples are shown for each time point. (−) value <50 pg/ml, (+) 50 pg/ml<value<500 pg/ml, (++) 500 pg/ml<value<5000 pg/ml, (+++) value >5000 pg/ml. There were no significant differences between the time points analyzed.
STEM_1067_sm_SuppFig3.tif123KSupplementary 3. Methylated DNA quantification assay of mature adipocytes, dedifferentiated adipocytes, SVF-derived MSCs and BM-derived MSCs. There was a significant difference between the mature adipocytes and the other studied cells, ** p<0.001.
STEM_1067_sm_SuppFig4.tif143KSupplementary 4. Mature adipocytes stimulated the growth of stimulated allogeneic lymphocytes while dedifferentiated adipocytes were able to inhibit their proliferation.
STEM_1067_sm_SuppFig5.tif2350KSupplementary 5. Dedifferentiated adipocytes supported the survival and complete differentiation of hematopoietic stem cells and formed colony-forming unit-granulocyte macrophages (CFU-GM) and burst-forming unit-erythroids (BFU-E).

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