察爾斯大夫 美麗殿堂 Dr. Charles Meridien Palace

Awakened by Cellular Stress：Isolation and Characterization of a Novel Population of Pluripotent Stem Cells Derived from Human Adipose Tissue（2）

Methods

Isolation of Muse-AT cells from Lipoaspirated Fat

Lipoaspirates (100–200 g per aspirate) were obtained from subcutaneous abdominal adipose of women undergoing elective liposuction. None of the investigators of this study had any contact with, nor any knowledge of any personal information relating to, these patients. Furthermore, human subjects were unidentifiable as well as all their characteristics and clinical records. Therefore, this study did not meet the criteria of human subjects research and HHS regulations did not apply (45 CFR 46.102(f)).

Lipoaspirate was repeatedly Washed with PBS until blood was completely removed from the tissue, and then incubated with equal volume of DMEM containing collagenase (0.1%, Sigma Aldrich) for 30 min at 37°C in a shaking incubator at 110 rpm, followed by incubation in 4°C, while still in collagenase and nutritionally deficient medium (no FCS), for 16 hours under severe hypoxia conditions. Digested material was then centrifuged at 1500 rpm for 10 minutes at 4°C. Supernatant containing adipose cell debris (dead adipocytes, macrophages, red blood cells, adipose stem cells among other cell components) was removed by aspiration and the remaining cell Pellets were washed several times with PBS. Pellets were re-suspended in PBS and incubated with a red blood cell lysis buffer (eBiosciences, San Diego, CA) for 10 min at R/T (2×). Remaining cell pellets containing cells highly resistant to severe cellular stress, were re-suspended in Dulbecco’s Modified Eagle Medium 1× (DMEM; CellGro, MediatechInc, Manassas, VA) comprised of 10% fetal bovine serum (FBS; Thermo Scientific Hyclone, Logan, UT) and 5% antibiotic-antimyocotic solution (CellGro, Mediatech Inc, Manassas, VA), and plated as cells in suspension as well as adherent cells. For ASC isolation, lipoaspirate material was subjected to collagenase digestion (0.1%, Sigma Aldrich) for 30 min at 37°C in a shaking incubator at 110 rpm, and ASCs were isolated and cultured as previously described.

Flow Cytometry Analysis

Floating Muse-AT cells were cultured in DMEM/10% FCS for 2 days followed by FACS analysis. Cells were washed in 2% inactivate FCS/0.05% sodium Azide/PBS and were re-suspended in 100 µl of the same buffer and incubated at 4°C for 1 hour in the presence or absence of primary unconjugated rat anti-human SSEA3 (EMD Millipore; Billerica, Massachusetts). Cells were then washed twice with the same buffer and incubated with the corresponding secondary FITC-conjugated anti-rat IgM (BD Biosciences; San Diego, CA) for 45 minutes at 4°C. After two consecutive washes, cells were incubated with PE-mouse anti-human CD105 (BD Biosciences, San Diego, CA) at 4°C for 1 hour. Cells were then washed and re-suspended in 200 µl of the same buffer. Analysis of count and cell type was performed using a FACS Calibur flow cytometer and cEllQuest Pro software.

Immunocytochemistry

Cells were fixed in 4% paraformaldehyde (20 min at R/T), washed in PBS, then incubated in 0.2% Triton for 20 min. After 2 successive washes in PBS, cells were blocked with 10% normal goat serum in 1% BSA solution for 60 min at R/T. Cells were then incubated with the primary antibodies overnight at 4°C. The following pluripotent stem cell markers were used: rat anti-human stage-specific embryonic antigen (SSEA3, Millipore, Billerica, MA), mouse anti-human octamer-binding transcription factor 3 and 4 (Oct3/4, Santa Cruz Biotech, Santa Cruz, CA), rabbit anti-human Nanog (Millipore, Billerica, MA), rabbit anti-human SRY-box 2 (Sox2, Millipore, Billerica, MA), and mouse anti-human TRA-1-60 (Abcam, Cambridge, MA); for mesenchymal cell lineages: rabbit anti-human preadipocyte factor 1 (Pref-1, [a.k.a. delta-like 1 homolog (drosophila), DLK1] preadipocyte marker, Santa Cruz Biotech, Santa Cruz, CA); mouse anti-human myosin D (MyoD, myocyte marker, R&D Systems, Minneapolis, MN), and mouse anti-human smooth muscle actin (SMA, myocyte marker, Thermo Scientific, Waltham MA); for endodermal cell lineages: mouse anti-human pan keratin (Santa Cruz, CA); rabbit anti-human α-fetoprotein (Dako, Santa Clara, CA); and mouse anti-human cytokeratin 7 (Millipore, Billerica, MA); and for ectodermal cell lineages: mouse anti-human neuron specific enolase (NSE, Millipore, Billerica, MA); rabbit anti-human glutamate receptor (Abcam, Cambridge, MA); rabbit anti-human NeuroD (Chemicon, Temecula CA); mouse anti-human nestin (Chemicon, Temecula CA); and rabbit anti-human microtubule-associated protein 2 (MAP2, AbDSerotech, Raleigh, NC). All primary antibodies were diluted 1:200 in PBS/0.1% BSA solution. Following treatment with primary antibodies, cells were washed 3 times with PBS and incubated for 1 hour at R/T with PBS/0.1% BSA containing secondary immunofluorescent antibodies (1:1000) Alexa Fluor 488 conjugated dye (mouse or rat, Invitrogen, Carlsbad, CA) or Texas Red conjugated dye (rabbit, Invitrogen, Carlsbad, CA). Cells were washed 4X with PBS and treated with PBS/0.2% DAPI for 10 minutes. Cells were then washed 3X with PBS. Images were acquired with an Evos immunofluorescence inverted microscope (Advanced Microscopy, Mill Creek, WA).

Induced Differentiation of Muse-ATs

Various differentiation media were used to induce differentiation of Muse cells-AT to the three germline cell lineages. For adipocyte formation, adherent Muse-AT cells were treated with adipogenic differentiation medium containing DMEM with 0.5 mM isobutylmethylxanthine, 1 µM dexamethasone, 10 µM insulin, 200 µM indomethacin and PPAR-γ (ZenBio, Inc, Research Triangle Park, NC) over 3 or 6 days at 37°C and 5% CO2. Adipocytes were detected using fluorescence lipid drop marker BODIPY-C16 (1:1000, Invitrogen, Carslbad, CA) following manufacturer specification.

For myocyte formation, adherent Muse-AT cells were incubated in DMEM containing with 10% FBS, 5% NHS, 50µM hydrocortisone, and 1% antibiotic-antimycotic solution over 3 or 6 days at 37°C and 5% CO2. Smooth muscle cells were identified by expression of smooth muscle actin (SMA) and skeletal muscle cells myosin D.

For hepatocyte and biliary cell induction, adherent Muse-AT cells were incubated in hepatocyte differentiation medium for 3 or 6 days, as previously described adherent Muse-AT cells were incubated in DMEM supplemented with 10% FBS, 10 µg/ml insulin, 5.5 µg/ml transferring, 6.7 ng/ml sodium selenite (ITS; Gibco, Life Technologies, Grand Island, NY), 10 nM dexamethasone (Sigma-Aldrich, St. Louis, MO), 100 ng/ml hepatocyte growth factor (HGF, Peprotech, Rocky Hill, NJ) and 50 ng/ml and fibroblast growth factor- 4 (FGF-4, R & D Systems, Minneapolis, MN) for 3 or 6 days. Hepatocytes were identified by immunohistochemistry using cytokeratin 7 and α-fetoprotein expression.

For neural cell formation, Muse cells-AT were incubated as non-adherent cells in ultra-low attachment plates (Corning Incorporated, Life Sciences, Manassas, VA) in the presence of neural differentiation medium 1 containing Neurobasal medium (Gibco, Life Technology, Grand Island, NY) supplemented with B-27 supplement serum free (Gibco, Life Technology, Grand Island, NY), 100 µg/ml kanamycin (Gibco, Life Technology, Grand Island, NY), 2 mM glutamine (Sigma-Aldrich, St. Louis, MO), 30 ng/ml bFGF (Peprotech, Rocky Hill, NJ) and 30 ng/ml EGF (Peprotech, Rocky Hill, NJ) for 7 days. Cells were then transferred to polystyrene culture slides (BD Biosciences, San Jose, CA) and cultured for another 7 days as adherent cells in the presence of neural differentiation medium 2 containing 1 DMEM supplemented with 2% FCS, 25 ng/ml bFGF and 25 ng/ml BDNF (Peprotech, Rocky Hill, NJ). Neural cells were identified by immunohistochemistry using nestin and MAP2 as indicated above.

Microarray Analysis

Muse-AT cells and ASCs were isolated from lipoaspirate material of three different patients. RNA was extracted using an RNeasy Mini Kit (Qiagen) and analyzed by Hokkaido System Science Co. Ltd. Array signals were processed and normalized using the GeneSpring GX version 12.1.0 (Agilent Technologies). Data has been deposited into the Gene Expression Omnibus databank with the access number GSE46353. The criteria for selecting differentially-expressed genes were preset as at least 2-fold difference in either direction plus statistical significance (P<0.05, unpaired t test). Microarray analysis was performed using the software program IPA via a license to Ingenuity (https://analysis.ingenuity.com/pa/login/​login.jsp) to identify (1) functional pathways (cell function, physiological function, diseases), (2) canonical signaling pathways (3) networks of related genes derived from genes changed in the analyzed comparisons and (4) upstream regulators. Further information regarding gene function was obtained from the program GeneDecks V3 at www.genecards.org. Statistical analyses were carried out by Fischer’s exact test (as performed automatically by the software). In determining which genes are only expressed in either Muse-ATs or ASCs, all samples, having been performed in triplicate, had to display uniform detection (indicated with at least 100 standard units) or absence (at most 30 standard units) along with a P-value <0.05.