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 treating diseases, the therapeutic effects are inconsistent across individuals⁶. However, the reason for MSC therapy instability is still unclear.

Molecular fluctuations, morphology, differentiation capacity, and therapeutic function comprise MSC heterogeneity. Some studies have also postulated that MSCs constitute subpopulations with different functions^7,8 and explored MSC heterogeneity via single-cell RNA sequencing (scRNA-seq)^9,10. The results revealed that human UC-MSCs have distinct subpopulations with specific transcriptomic features, whereas few studies have successfully isolated so-called MSC subpopulations. We previously dissected human UC-MSCs into three subgroups according to their signatures via scRNA-seq and bioinformatics analysis, in which the BAMBI^highMFGE8^high UC-MSC subpopulation was further purified and functionally tested¹¹. However, this subgroup failed to alleviate lupus nephritis. Thus, it is necessary to test the therapeutic effects of BAMBI^highMFGE8^high MSCs in other disorders to understand their authentic functions.

This protocol describes methods for isolating the BAMBI^highMFGE8^high subgroup from human UC-MSCs via fluorescence-activated cell sorting (FACS) by flow cytometry and the characteristics of the BAMBI^highMFGE8^high subgroup.

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 previously reported¹¹.

1. UC-MSC culture and identification before isolation

1. Once the MSCs reach 70-80% confluence (P0), wash the cells once with PBS and add 1 mL of 0.25% trypsin-EDTA for 2 min at 37 °C. Then, add 9 mL of complete medium to neutralize the trypsin, transfer the cell suspension to a 15 mL centrifuge tube, and centrifuge at 300 × g for 5 min to collect the cells. Discard the supernatant and add the appropriate complete medium to resuspend the cells. Transfer the cells in each dish to three T75 flasks (P1) and culture in a cell incubator at 37 °C and 5% CO₂.
2. After passaging the UC-MSCs 2x via trypsinization, at P3, harvest the cells via the same steps described in step 1.1. After centrifugation, discard the supernatant. Resuspend the cells in FACS staining buffer (1x PBS containing 2% FBS) and count them. Add FACS staining buffer to a final concentration of 5-10 × 10⁶ cells/mL and keep the cell suspension on ice.
3. Distribute 100 µL per tube of this cell suspension into different 1.5 mL tubes. Add isotype controls and FACS antibodies against CD29, CD73, CD90, CD105, CD14, CD34, CD45, CD79, and HLA-DR (all at 1:200) to the cells at 4 °C for 30 min.
4. Wash the cells 2x with the FACS staining buffer and centrifuge at 300 × g for 5 min. Discard the supernatant and resuspend the precipitates in 200 µL of FACS staining buffer for flow cytometry analysis to identify MSC markers.

2. Isolation of BAMBI^high MFGE8^high UC-MSCs by flow cytometry

1. Culture UC-MSC to a density of approximately 5-10 × 10⁶ cells when isolating BAMBI^highMFGE8^high UC-MSCs. Dissociate the cells with 0.5 mM EDTA for 5 min until they start to have a round morphology, add the complete medium to transfer the cells to a 15 mL conical tube, and pipet the cell suspension up and down several times to prepare a single-cell suspension. Use 10 µL of the cell suspension to count the cells and calculate the total number of cells harvested. Centrifuge the conical tube with the cell suspension at 300 × g for 5 min to collect the cells.
2. Resuspend the cells in 1 mL of complete medium to a final concentration of 5-10 × 10⁶ cells/mL and keep the cell suspension on ice.
3. Divide the cells into four 1.5 mL microcentrifuge tubes (blank cells only; BAMBI-labeled cells; MFGE8-labeled cells; and both BAMBI- and MFGE8-labeled cells).
4. Add primary antibodies at appropriate concentrations (MFGE8 and BAMBI, both at 1:100) to the tubes, mix, and incubate the cells at room temperature for 15 min.
5. Wash the labeled cells once with 1x PBS, and centrifuge at 300×g for 5 min. Discard the supernatant. Resuspend the cells in 1 mL of complete medium, add conjugated fluorescent secondary antibodies (goat anti-rabbit IgG H&L Alexa Fluor 488 and Alexa Fluor 647, all at 1: 1,000) to the cells, mix, and incubate the cells at room temperature for 15 min in the dark.
6. Wash the labeled cells once with 1x PBS, as described in step 2.5. Resuspend the cells in 500 µL of complete medium, filter through a 70 µm cell strainer to eliminate clumps and debris, and transfer the filtrate into a 15 mL tube for flow cytometry sorting.
7. Run the blank cell tube without adding an antibody (negative control) and adjust the forward scatter (FSC) and side scatter (SSC) to gate the scale of the negative population with antibody staining.
8. Run the single antibody-labeled cell tubes (i.e., MFGE8 + goat anti-rabbit IgG H&L Alexa Fluor 488, or BAMBI + goat anti-rabbit IgG H&L Alexa Fluor 647) as a gating control to determine where the positivity starts in the plot.
9. Run the experimental sample tube(s) to sort and collect the BAMBI^highMFGE8^high cell population.
10. Plate the sorted BAMBI^highMFGE8^high MSCs in a 24-well plate and cultivate the cells in a cell incubator at 37 °C and 5% CO₂.
11. When the sorted BAMBI^highMFGE8^high MSCs have grown for two passages to obtain enough cells, dissociate the cells and perform a post sorting analysis to ensure the purity of the sorted cell populations by flow cytometry, as described in steps 2.1-2.9. Alternatively, examine the purity of the sorted cells with conventional immunofluorescence against human BAMBI and MFGE8 expression if the number of collected cells is too small.

3. Characterization of the BAMBI^high MFGE8^high MSCs

1. Grow the sorted BAMBI^highMFGE8^high MSCs in a 12-well plate, examine their cell morphology under a microscope, and compare them with that of unsorted MSCs.
   NOTE: In general, BAMBI^highMFGE8^high cells grow faster than unsorted UC-MSCs.
2. When the cells are approximately 90% confluent in the 12-well plate, aspirate the cell culture medium, wash the cells once with 1x PBS, and add 500 µL of RNA extraction reagent to the cells. Shake the plate slowly at room temperature (RT) for 5 min.
3. Pipette the mixture and transfer it to an RNase- and DNase-free tube. Add 100 µL of chloroform, cap the tube, and shake the mixture vigorously by vortexing for 15 s. Then, allow the tube to stand at RT for 3 min.
4. Centrifuge the tube for 15 min at 11,000 × g and 4 °C.
5. Transfer approximately 200 µL of the upper aqueous layer into a new tube. Add the same volume of isopropanol and pipette thoroughly up and down several times. Then, allow the mixture to stand at RT for 10 min.
6. Centrifuge for 15 min at 11,000 × g and 4 °C. Discard the supernatant.
7. Add 500 µL of 75% ethanol to the pellet in the tube and invert the tube gently several times.
8. Centrifuge the tube for 5 min at 6,000 × g and 4 °C. Discard the supernatant. Repeat the centrifugation once for 10 s at 6,000 × g, carefully aspirate the remaining fluid via pipette, and dry the tube at RT.
9. Add 10 µL of RNase-free water to dissolve the RNA pellet, and measure the RNA concentration using a spectrophotometer.
10. Follow the manufacturer's instructions to synthesize first-strand cDNA from up to 1 µg of total RNA.
11. Use a qPCR kit to detect and quantify gene expression, according to the manufacturer's instructions.
    NOTE: The sequences of the DNA primers used are listed in Table 1.

Results

Figure 1 shows the cell surface marker expression profiles of human UC-MSCs. Cultured MSCs were strongly positive for CD44, CD73, CD90, and CD105 expression and negative for CD14, CD34, CD45, CD79, and HLA-DR expression. The BAMBI^highMFGE8^high MSCs were sorted from cultivated human UC-MSCs, and their BAMBI and MFGE8 expression was reanalyzed by flow cytometry after expansion for 3-4 passages (Figure 2). In this process, the frequency o...

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