Isolation of Human BAMBIhighMFGE8high Umbilical Cord-Derived Mesenchymal Stromal Cells

Summary

The isolation of BAMBI^highMFGE8^high MSCs, one of the three main subgroups constituting heterogeneous human UC-MSCs, is helpful for fully understanding the characteristics and functions of this subtype for its future application to improve clinical efficacy in specific diseases. Here, we present a method for sorting BAMBI^highMFGE8^high UC-MSCs.

Abstract

Umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) present low immunogenicity and potent immunomodulatory effects for treating various diseases. Human UC-MSCs are a heterogeneous population consisting of three main subpopulations with different cell shapes, proliferation rates, differentiation abilities, and immune regulatory functions. Previously, BAMBI^highMFGE8^high UC-MSCs, the first subgroup successfully isolated from UC-MSCs were found to fail to alleviate lupus nephritis. Hence, the function and underlying mechanism of this subgroup in MSC therapy for diseases remains unknown. It is necessary to isolate and further investigate BAMBI^highMFGE8^high UC-MSCs in terms of their phenotype, metabolism, and function to completely understand the nature of this MSC subgroup. In this protocol, we describe a detailed method for isolating the BAMBI^highMFGE8^high subpopulation from human UC-MSCs. The subpopulation of UC-MSCs is labeled with two surface markers, BAMBI and MFGE8, by flow cytometry sorting. The isolated cells are cultured and verified by flow cytometry analysis. The specific genes expressed in the BAMBI^highMFGE8^high UC-MSCs are identified by RT-qPCR. This protocol results in highly efficient and pure cell sorting and describes the marker profiles of the BAMBI^highMFGE8^high UC-MSCs.

Introduction

Human mesenchymal stromal/stem cells (MSCs) are somatic progenitors capable of differentiating into osteocytes, adipocytes, chondrocytes, and other cell types¹. MSCs were first isolated from bone marrow and are widely derived from the umbilical cord, adipose tissue, and other tissues². Because UC-MSCs are easily obtained and exhibit low immunogenicity and immunosuppressive effects, they are widely applied in clinical trials to treat various diseases^3,4,5. Although MSC therapy shows promising potential for t....

Protocol

This study was conducted in accordance with the principles set forth under the 1989 Declaration of Helsinki and approved by the Ethics Committee at the Affiliated Drum Tower Hospital of Nanjing University Medical School (approval number: 202019701). Human umbilical cords were obtained from healthy mothers at the Affiliated Drum Tower Hospital of Nanjing University Medical School after natural labor, who gave their informed consent for their use in this work. Primary UC-MSCs were isolated from human umbilical cords as pre.......

Discussion

This protocol describes how to isolate and enrich the BAMBI^highMFGE8^high subpopulation from human UC-MSCs. The method is important for further study of the morphology, growth, and function of this MSC subgroup. Some steps are vital for the successful isolation and high yield of BAMBI^highMFGE8^high cells.

First, the most critical technical aspect to consider is the use of an appropriate cell dissociation solution in the present protocol. Although conve.......

Materials

Name	Company	Catalog Number	Comments
0.6 mL microcentrifuge tube	Corning	Axygen MCT-060-A
1.5 mL microcentrifuge tubes	Beijing Labgic Technology	MCT-001-150
100 mm cell culture dish	Beijing Labgic Technology	12311
12 well plate	Beijing Labgic Technology	11210
15 mL centrifuge tube	Nanjing Vazyme Material Technology	TCF00115
24 well plate	Beijing Labgic Technology	11310
50 mL centrifuge tube	Nanjing Vazyme Material Technology	TCF00150
5 mL Round-Bottom Tubes	Corning	FALCON 352003
70 μm cell strainer	Falcon	352350	Dilution: 1:1000
APC anti-human CD79a (Igα) Antibody	BioLegend	333505	581 Dilution: 1:200
APC/Cyanine7 anti-human CD73 (Ecto-5'-nucleotidase) Antibody	BioLegend	344022	G46-6 Dilution: 1:200
APC-Cy7 Mouse IgG1, κ Isotype Control	BD Bioscience	557873	MOPC-31C (Isotype Control) Dilution: 1:200
BAMBI antibody	Bioss	bs-12418R
Brilliant Violet 510 anti-mouse/human CD44 Antibody	BioLegend	103044	5E10 Dilution: 1:200
Brilliant Violet 510 Rat IgG2b, κ Isotype Ctrl Antibody	BioLegend	400646	MOPC-21 (Isotype Control) Dilution: 1:200
CD105 (Endoglin) Monoclonal Antibody  APC	eBioscience	17-1057-42	HM47 Dilution: 1:200
Cell Counting Chamber Slides	Shanghai QIUJING	XB-K-25
Centrifuge	Beijing BAIYANG	BY-320C
ChamQ Universal SYBR qPCR Master Mix	Vazyme	Q711-02
Chloroform	XILONG Scientific	13700908
DMEM/F-12 (1:1) basic (1x)	Gibco	C11330500BT
EDTA (0.5 M), pH 8.0, Rnase free	Invitrogen	AM9260G	Dilution: 1:1000
Ethanol	XILONG Scientific	12803405
Fetal Bovine Serum (FBS)	Gibco	10099-141C
FITC Mouse Anti-Human CD34	BD Bioscience	555821	IM7 Dilution: 1:200
FITC Mouse Anti-Human CD45	BD Bioscience	555482	AD2 Dilution: 1:200
FITC Mouse Anti-Human HLA-DR	BD Bioscience	555811	SN6 Dilution: 1:200
Flow Cytometer	BD Bioscience	FACSAria™ III Cell SorterAria
Flowjo	BD Bioscience	V10
Gentle Cell Dissociation Reagent	STEMCELL Technologies	100-0485
Goat Anti-Mouse IgG H&L (Alexa Fluor 488)	abcam	ab150113	HI30 Dilution: 1:200
Goat Anti-Rabbit IgG H&L (AlexaFluor 594)	abcam	ab150080	Dilution: 1:1000
HiScript II Q RT SuperMix for qPCR (+gDNA wiper)	Vazyme	R223-01
Inverted Microscopes	Nikon	ECLIPSE Ts2
Isopropyl Alcohol	XILONG Scientific	12802505
MFGE8 antibody	Biorbyt	orb388429	Dilution: 1:100
Microcentrifuge	Thermo Fisher Scientific	FRESCO 21
Mouse IgG1 kappa Isotype Control APC	eBioscience	17-4714-42	P3.6.2.8.1 (Isotype Control) Dilution: 1:200
Mouse IgG1 kappa Isotype Control FITC	eBioscience	11-4714-42	eBMG2b (Isotype Control) Dilution: 1:200
Mouse IgG2b kappa Isotype Control FITC	eBioscience	11-4732-42	RTK4530 (Isotype Control) Dilution: 1:200
PBS (10x)	Sangon Biotech (Shanghai)	E607016-0500
PE-Cy5 Mouse Anti-Human CD90	BD Bioscience	555597	P3.6.2.8.1 (Isotype Control) Dilution: 1:200
PE-Cy5 Mouse IgG1 κ Isotype Control	BD Bioscience	550618
Penicillin-Streptomycin 100x	Cytiva	SV30010	Dilution: 1:100
Real-Time PCR System	Applied Biosystems byThermo Fisher Scientific	Q6
RNase-free water	QIAGEN	129112
Spectrophotometer	Thermo Fisher Scientific	NanoDrop One(840-317400)
Sterile micropipette tips	Beijing Labgic Technology		Dilution: 1:100
T75 cell culture flask	Beijing Labgic Technology	13212A
Thermal Cycler	Applied Biosystems byThermo Fisher Scientific	Veriti
Tri reagent	Sigma Aldrich	T9424
Typsin-EDTA Solution	Bio-Channel Biotechnology	BC-CE-005
Water-Jacketed CO2  Incubator	Thermo Fisher Scientific	3111