Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

Podsumowanie

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 Sca1^high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

Streszczenie

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 function^1,2. 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 resistance^3,4. 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 Sca1^high ASCs.

Wprowadzenie

Stem cell antigen 1 (Sca1, or Ly6A/E) was first identified as a cell surface marker expressed by hematopoietic and mesenchymal stem cells^5,6. The stromal vascular fraction (SVF) of adipose tissue obtained from mouse fat depots is a heterogeneous population of cells comprising of fibroblasts, macrophages, vascular endothelial cells, neuronal cells, and adipocyte progenitor cells⁷. Adipocyte progenitor cells, or adipose-derived stem cells (ASCs) are non-lipid-laden cells that reside in the collagen-rich perivascular extracellular matrix (ECM)⁸. Approximately 50% of the SVF consist of ASCs, which are characterized as lineage-negative (Lin....

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Protokół

Ethics Statement: The University of Michigan Committee on Use and Care of Animals (UCUCA) has approved all methods and protocols in accordance with the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, National Research Council). Mice are maintained in a University of Michigan vivarium and are given free access to food and water and kept on a 12 hr dark/light cycle.

1. Preparations

1. Prepare primary culture media with DMEM, 10% FBS, 1x P/S/G, and 1x antibiotics/antifungals. This will be used to keep tissues in before digestion. Place stock and aliquot in 37 ºC water bath.
2. Pr....

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Wyniki

Enrichment of Sca1^high ASCs from Different Fat Pads.

The vascular stromal cells isolated from SQ fat display fibroblast-like, stretched cell shape regardless of Sca1 expression level (Figure 1A). On the other hand, VIS (eWAT-derived) Sca1^high and Sca1^low cells demonstrate distinct difference in their cell shape. Like SQ (iWAT-derived) Sca1^high cells, VIS (eWAT-derived).......

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Dyskusje

Herein we demonstrate the isolation and immunomagnetic cell separation of murine ASCs from different fat pads and their use for in vitro experiments. The presented method is effective for the quick isolation of large number of Sca1-positive ASCs, which is advantageous over the technically complex and expensive FACS-mediated isolation of ASCs^9,14. Unlike FACS, immunomagnetic cell separation does not allow the use of multiple antigen for the identification of a target cell population. Nonetheless, if th.......

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Materiały

Name	Company	Catalog Number	Comments
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
60 mm 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 50 ml 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
1 M HEPES	Gibco	15630-080
0.34 N NaOH	Prepare in house
Cover slips	Corning	2870-22
Alexa Fluor 594 carboxylic acid, succinimidyl ester, mixed isomers	Invitrogen	A-20004
0.89 M NaHCO3	Gibco	25080-094