Immunomagnetisk Adskillelse af Fat Depot-specifikke Sca1<sup> høj</sup> adipøst afledte stamceller (ASC'er)
Summary
We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.
Abstract
The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.
Introduction
Stem cell antigen 1 (Sca1 eller Ly6A / E) blev først identificeret som en celle overflade markør udtrykt af hæmatopoietiske og mesenkymale stamceller 5,6. Den stromale vaskulære fraktion (SVF) af fedtvæv opnået fra mus fedtdepoter er en heterogen population af celler omfattende af fibroblaster, makrofager, vaskulære endotelceller, neuronale celler og adipocyt progenitorceller 7. Adipocyt progenitorceller eller adipøst afledte stamceller (ASC) er ikke-lipid-laden celler, der bor i kollagen-rige perivaskulær ekstracellulær matrix (ECM) 8. Ca. 50% af SVF bestå af ASC'er, som er karakteriseret som slægt-negative (Lin -) og CD29 +: CD34 +: Sca1 + 9. De fleste af disse celler er Sca1 +: CD24 – adipocyt progenitorer, som er i stand adipocytdifferentiering in vitro; dog kun en brøkdel af celler (0,08% af SVF) udgør Sca1 <sup> +: CD24 + celler, der er fuldt ud i stand til at prolifererende og differentiere til adipocyter i in vivo betingelser 9. På trods af den potentielle forbehold for at bruge Sca1 + SVF uden at diskriminere CD24 + celler fra CD24 – celler, isolere Sca1 + ASC'er fra fedtdepoter hjælp immunomagnetisk celleseparation er en effektiv og praktisk tilgang til at bestemme celle-autonome fænotype af primær adipocyt stamceller.
Inden for fedme og diabetes, væv fibrose og inflammation spiller en kritisk rolle i udviklingen og vedligeholdelsen af type-2 diabetes 3. For nylig, Tokunaga et al. viste, at Sca1 høje celler isoleret fra lyskebrok (eller subkutan, SQ) og perigonadal (eller visceral, VIS) C57BL6 / J fedtdepoter udviser forskellige gen signaturer og ECM remodellering in vitro 10. MMP14 (MT1-MMP), en prototypisk medlem af membranen-type matrixmetalloproteinase (MMP) familien medierer udviklingen af hvide fedtvæv (WAT) gennem sin kollagenolytisk aktivitet 1.
Eksempler på eksperimenter, der kan udføres med celler isoleret og beriget gennem følgende protokol omfatter tre-dimensionelle kultur, differentiering undersøgelser, collagen nedbrydning analyser, og RNA-sekventering 10,11. Nedbrydning analyser bør foretages med syre ekstraheret kollagen for at sikre bevarelsen af telopeptidet 11,12. Følgende protokol vil vise de metoder til isolering primære vaskulære stromaceller fra forskellige fedtdepoter og berige adipocyt progenitorceller ved anvendelse af immunmagnetisk celleseparation. Gyldigheden af cellen sortering vil blive vurderet med flowcytometri og ved at bruge Sca1-GFP mus, der udtrykker GFP i Sca1 + celler, drevet af en Sca1 promotor 13.
Protocol
Representative Results
Discussion
Heri vi demonstrere isolation og immunomagnetisk celle adskillelse af murine ASC'er fra forskellige fedtpuder og deres anvendelse til in vitro-forsøg. I denne procedure er effektiv til hurtig isolering af store antal Sca1-positive ASC'er, hvilket er fordelagtigt over teknisk komplicerede og dyre FACS-medieret isolering af ASC'er 9,14. I modsætning FACS, er immunomagnetisk celleseparation ikke tillade anvendelse af multiple antigen til identifikation af en målcellepopulation. Men hvis o…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde er støttet af NIH DK095137 (til THC). Vi takker de nuværende og tidligere lab medlemmer der har bidraget til udviklingen og kvaliteten af de beskrevne metoder.
Materials
| Type 3 Collagenase | Worthington Biochemical | LS004182 | Tissue digestion |
| DMEM | Gibco | 11965-092 | High-glucose culture medium |
| Pen/Strep/Glutamine (100x) | Gibco | 10378-016 | Media antibiotic |
| Anti-anti (100x) | Gibco | 15240-062 | Media antifungal |
| FBS | Gibco | 16000-044 | |
| PBS (1x, pH 7.4) | Gibco | 10010-023 | |
| Trypsin (0.05%) | Gibco | 25300-054 | |
| Cell strainer | BD Bioscience | 352360 | 100-μm cell strainer |
| 60mm plates | BD Falcon | 353004 | |
| Scissors | FST | 14001-12 | Large |
| Scissors | FST | 14091-11 | Fine, curved tip |
| Large Forceps | FST | 11000-12 | |
| Fine Forceps | Any vendor | ||
| 25G 5/8” needles | BD | 305122 | |
| 22G 1.5” needles | BD | 305159 | |
| 15 ml conical tubes | BD Falcon | 352097 | |
| 50 ml conical tubes | BD Falcon | 352098 | |
| MACS separation columns | Miltenyi Biotec | 130-042-201 | |
| Anti-Sca1 microbead kit (FITC) | Miltenyi Biotec | 130-092-529 | FITC-anti-Sca1 1ºAb and anti-FITC microbeads 2ºAb |
| AutoMACS running buffer | Miltenyi Biotec | 130-091-221 | |
| MiniMACS separator | Miltenyi Biotec | 130-042-102 | |
| MACS MultiStand | Miltenyi Biotec | 130-042-303 | |
| Blue chux pads | Fisher | 276-12424 | |
| Absorbent pads | Fisher | 19-165-621 | |
| Styrofoam board | Use from 50ml tubes | ||
| 70% ethanol | |||
| Isoflurane | Any vendor | ||
| Rat IgG2a Alexa Fluor 647 | Invitrogen | R2a21 | |
| Rat IgG2a anti-mouse Sca1 Alexa Fluor 647 | Invitrogen | MSCA21 | |
| Rat IgG2a R-PE | Invitrogen | R2a04 | |
| Rat IgG2a anti-mouse F4/80 R-PE | Invitrogen | MF48004 | |
| Round-bottom tube | BD Falcon | 352058 | |
| HBSS (–Ca, –Mg) | Gibco | 14175-095 | |
| HBSS (+Ca, +Mg) | Gibco | 14025-092 | For collagenase solution |
| Type I collagen (2.7 mg/ml in 37mm acetic acid | Prepare in house12 | ||
| 10x MEM | Gibco | 11430-030 | |
| 1M HEPES | Gibco | 15630-080 | |
| 0.34N NaOH | Prepare in house | ||
| Cover slips | Corning | 2870-22 | |
| Alexa Fluor 594 carboxylic acid, succinimidyl ester, mixed isomers | Invitrogen | A-20004 | |
| 0.89M NaHCO3 | Gibco | 25080-094 |
References
- Chun, T. H., et al. A pericellular collagenase directs the 3-dimensional development of white adipose tissue. Cell. 125 (3), 577-591 (2006).
- Chun, T. H., et al. Genetic link between obesity and MMP14-dependent adipogenic collagen turnover. Diabetes. 59 (10), 2484-2494 (2010).
- Chun, T. H. Peri-adipocyte ECM remodeling in obesity and adipose tissue fibrosis. Adipocyte. 1 (2), 89-95 (2012).
- Sun, K., Tordjman, J., Clement, K., Scherer, P. E. Fibrosis and adipose tissue dysfunction. Cell Metab. 18 (4), 470-477 (2013).
- Spangrude, G., Heimfeld, S., Weissman, I. Purification and Characterization of Mouse Hematopoietic Stem Cells. Science. 241, 58-62 (1988).
- Welm, B. E., et al. Sca-1(pos) cells in the mouse mammary gland represent an enriched progenitor cell population. Dev Biol. 245 (1), 42-56 (2002).
- Gesta, S., Tseng, Y. H., Kahn, C. R. Developmental origin of fat: tracking obesity to its source. Cell. 131 (2), 242-256 (2007).
- Tang, W., Zeve, D., Suh, J. M., Bosnakovski, D., Kyba, M., Hammer, R. E., Tallquist, M. D., Graff, J. M. White fat progenitor cells reside in the adipose vasculature. Science. 322, 583-586 (2008).
- Rodeheffer, M. S., Birsoy, K., Friedman, J. M. Identification of white adipocyte progenitor cells in vivo. Cell. 135 (2), 240-249 (2008).
- Tokunaga, M., et al. Fat depot-specific gene signature and ECM remodeling of Sca1(high) adipose-derived stem cells. Matrix Biol. 36, 28-38 (2014).
- Chun, T. H., Inoue, M. 3-D adipocyte differentiation and peri-adipocyte collagen turnover. Methods Enzymol. 538, 15-34 (2014).
- Rajan, N., Habermehl, J., Cote, M. F., Doillon, C. J., Mantovani, D. Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications. Nat Protoc. 1 (6), 2753-2758 (2006).
- Ma, X., Robin, C., Ottersbach, K., Dzierzak, E. The Ly-6A (Sca-1) GFP Transgene is Expressed in all Adult Mouse Hematopoietic Stem Cells. Stem Cells. 20 (6), 514-521 (2002).
- Berry, R., Rodeheffer, M. S. Characterization of the adipocyte cellular lineage in vivo. Nat Cell Biol. 15 (3), 302-308 (2013).
- Jeffery, E., Church, C. D., Holtrup, B., Colman, L., Rodeheffer, M. S. Rapid depot-specific activation of adipocyte precursor cells at the onset of obesity. Nat Cell Biol. 17 (4), 376-385 (2015).
- Mori, S., Kiuchi, S., Ouchi, A., Hase, T., Murase, T. Characteristic Expression of Extracellular Matrix in Subcutaneous Adipose Tissue Development and Adipogenesis; Comparison with Visceral Adipose Tissue. Int J Biol Sci. 10 (8), 825-833 (2014).
- Ong, W. K., et al. Identification of Specific Cell-Surface Markers of Adipose-Derived Stem Cells from Subcutaneous and Visceral Fat Depots. Stem Cell Reports. 2 (2), 171-179 (2014).
