Name: Isolation of Human BAMBIhighMFGE8high Umbilical Cord-Derived Mesenchymal Stromal Cells (Video) | JoVE Journal
Uploaded: 2025-01-10T14:20:00.000+00:00
Duration: 6 min 5 s
Description: Learn to isolate BAMBI high MFGE8 high human UC-MSCs using flow cytometry and single-cell transcriptomic analysis. This method reveals distinct phenotypes and supports selecting optimal subgroups for treating lupus nephritis.

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5 minutes

This work was supported by the National Natural Science Foundation of China (grant no. 82271843).

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
<ol>
	<li>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 &#176;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 &#215; 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 &#176;C and 5% CO2.</li>
	<li>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 &#215; 106 cells/mL and keep the cell suspension on ice.</li>
	<li>Distribute 100 &#181;L 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 &#176;C for 30 min.</li>
	<li>Wash the cells 2x with the FACS staining buffer and centrifuge at 300 &#215; g for 5 min. Discard the supernatant and resuspend the precipitates in 200 &#181;L of FACS staining buffer for flow cytometry analysis to identify MSC markers.</li>
</ol>
2. Isolation of BAMBIhigh MFGE8high UC-MSCs by flow cytometry
<ol>
	<li>Culture UC-MSC to a density of approximately 5-10 &#215; 106 cells when isolating BAMBIhighMFGE8high 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 &#181;L 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 &#215; g for 5 min to collect the cells.</li>
	<li>Resuspend the cells in 1 mL of complete medium to a final concentration of 5-10 &#215; 106 cells/mL and keep the cell suspension on ice.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Wash the labeled cells once with 1x PBS, and centrifuge at 300&#215;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&#38;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.</li>
	<li>Wash the labeled cells once with 1x PBS, as described in step 2.5. Resuspend the cells in 500 &#181;L of complete medium, filter through a 70 &#181;m cell strainer to eliminate clumps and debris, and transfer the filtrate into a 15 mL tube for flow cytometry sorting.</li>
	<li>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.</li>
	<li>Run the single antibody-labeled cell tubes (i.e., MFGE8 + goat anti-rabbit IgG H&#38;L Alexa Fluor 488, or BAMBI + goat anti-rabbit IgG H&#38;L Alexa Fluor 647) as a gating control to determine where the positivity starts in the plot.</li>
	<li>Run the experimental sample tube(s) to sort and collect the BAMBIhighMFGE8high cell population.</li>
	<li>Plate the sorted BAMBIhighMFGE8high MSCs in a 24-well plate and cultivate the cells in a cell incubator at 37 &#176;C and 5% CO2.</li>
	<li>When the sorted BAMBIhighMFGE8high 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.</li>
</ol>
3. Characterization of the BAMBIhigh MFGE8high MSCs
<ol>
	<li>Grow the sorted BAMBIhighMFGE8high MSCs in a 12-well plate, examine their cell morphology under a microscope, and compare them with that of unsorted MSCs. 
	NOTE: In general, BAMBIhighMFGE8high cells grow faster than unsorted UC-MSCs.</li>
	<li>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 &#181;L of RNA extraction reagent to the cells. Shake the plate slowly at room temperature (RT) for 5 min.</li>
	<li>Pipette the mixture and transfer it to an RNase- and DNase-free tube. Add 100 &#181;L 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.</li>
	<li>Centrifuge the tube for 15 min at 11,000 &#215; g and 4 &#176;C.</li>
	<li>Transfer approximately 200 &#181;L 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.</li>
	<li>Centrifuge for 15 min at 11,000 &#215; g and 4 &#176;C. Discard the supernatant.</li>
	<li>Add 500 &#181;L of 75% ethanol to the pellet in the tube and invert the tube gently several times.</li>
	<li>Centrifuge the tube for 5 min at 6,000 &#215; g and 4 &#176;C. Discard the supernatant. Repeat the centrifugation once for 10 s at 6,000 &#215; g, carefully aspirate the remaining fluid via pipette, and dry the tube at RT.</li>
	<li>Add 10 &#181;L of RNase-free water to dissolve the RNA pellet, and measure the RNA concentration using a spectrophotometer.</li>
	<li>Follow the manufacturer&#39;s instructions to synthesize first-strand cDNA from up to 1 &#181;g of total RNA.</li>
	<li>Use a qPCR kit to detect and quantify gene expression, according to the manufacturer&#39;s instructions. 
	NOTE: The sequences of the DNA primers used are listed in Table 1.</li>
</ol>

Functional Analysis of BAMBIhighMFGE8high UC-MSC Subpopulations

<ol><li>Liechty, K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+mesenchymal+stem+cells+engraft+and+demonstrate+site-specific+differentiation+after+in+utero+transplantation+in+sheep%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep.</a> Nat Med. 6 (11), 1282-1286 (2000).</li>
<li>Zhou, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Challenges+and+advances+in+clinical+applications+of+mesenchymal+stromal+cells%5BTitle%5D)+AND+%22J+Hematol+Oncol%22%5BJournal%5D)">Challenges and advances in clinical applications of mesenchymal stromal cells.</a> J Hematol Oncol. 14 (1), 24(2021).</li>
<li>Xie, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tnf-alpha-mediated+m%286%29a+modification+of+elmo1+triggers+directional+migration+of+mesenchymal+stem+cell+in+ankylosing+spondylitis%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Tnf-alpha-mediated m(6)a modification of elmo1 triggers directional migration of mesenchymal stem cell in ankylosing spondylitis.</a> Nat Commun. 12 (1), 5373(2021).</li>
<li>Mcguire, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem+cell-derived+interleukin-28+drives+the+selection+of+apoptosis+resistant+bone+metastatic+prostate+cancer%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Mesenchymal stem cell-derived interleukin-28 drives the selection of apoptosis resistant bone metastatic prostate cancer.</a> Nat Commun. 12 (1), 723(2021).</li>
<li>Yuan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem+cell+therapy+induces+flt3l+and+cd1c%28%2B%29+dendritic+cells+in+systemic+lupus+erythematosus+patients%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Mesenchymal stem cell therapy induces flt3l and cd1c(+) dendritic cells in systemic lupus erythematosus patients.</a> Nat Commun. 10 (1), 2498(2019).</li>
<li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Umbilical+cord+mesenchymal+stem+cell+transplantation+in+active+and+refractory+systemic+lupus+erythematosus%3A+A+multicenter+clinical+study%5BTitle%5D)+AND+%22Arthritis+Res+Ther%22%5BJournal%5D)">Umbilical cord mesenchymal stem cell transplantation in active and refractory systemic lupus erythematosus: A multicenter clinical study.</a> Arthritis Res Ther. 16 (2), R79(2014).</li>
<li>Ortiz, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem+cell+engraftment+in+lung+is+enhanced+in+response+to+bleomycin+exposure+and+ameliorates+its+fibrotic+effects%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects.</a> Proc Natl Acad Sci U S A. 100 (14), 8407-8411 (2003).</li>
<li>Phinney, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+heterogeneity+of+mesenchymal+stem+cells%3A+Implications+for+cell+therapy%5BTitle%5D)+AND+%22J+Cell+Biochem%22%5BJournal%5D)">Functional heterogeneity of mesenchymal stem cells: Implications for cell therapy.</a> J Cell Biochem. 113 (9), 2806-2812 (2012).</li>
<li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+transcriptome+atlas+of+human+mesenchymal+stem+cells+exploring+cellular+heterogeneity%5BTitle%5D)+AND+%22Clin+Transl+Med%22%5BJournal%5D)">Single-cell transcriptome atlas of human mesenchymal stem cells exploring cellular heterogeneity.</a> Clin Transl Med. 11 (12), e650(2021).</li>
<li>Chen, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+and+spatial+transcriptomics+decodes+wharton%27s+jelly-derived+mesenchymal+stem+cells+heterogeneity+and+a+subpopulation+with+wound+repair+signatures%5BTitle%5D)+AND+%22Adv+Sci+%28Weinh%29%22%5BJournal%5D)">Single-cell and spatial transcriptomics decodes wharton's jelly-derived mesenchymal stem cells heterogeneity and a subpopulation with wound repair signatures.</a> Adv Sci (Weinh). 10 (4), e2204786(2023).</li>
<li>Chen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dissecting+heterogeneity+reveals+a+unique+bambi%28high%29+mfge8%28high%29+subpopulation+of+human+uc-mscs%5BTitle%5D)+AND+%22Adv+Sci+%28Weinh%29%22%5BJournal%5D)">Dissecting heterogeneity reveals a unique bambi(high) mfge8(high) subpopulation of human uc-mscs.</a> Adv Sci (Weinh). 10 (1), e2202510(2022).</li>
<li>Tsuji, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+different+cell-detaching+methods+on+the+viability+and+cell+surface+antigen+expression+of+synovial+mesenchymal+stem+cells%5BTitle%5D)+AND+%22Cell+Transplant%22%5BJournal%5D)">Effects of different cell-detaching methods on the viability and cell surface antigen expression of synovial mesenchymal stem cells.</a> Cell Transplant. 26 (6), 1089-1102 (2017).</li>
<li>Lai, T. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Different+methods+of+detaching+adherent+cells+and+their+effects+on+the+cell+surface+expression+of+fas+receptor+and+fas+ligand%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Different methods of detaching adherent cells and their effects on the cell surface expression of fas receptor and fas ligand.</a> Sci Rep. 12 (1), 5713(2022).</li>
<li>Avinash, K., Malaippan, S., Dooraiswamy, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+of+isolation+and+characterization+of+stem+cells+from+different+regions+of+oral+cavity+using+markers%3A+A+systematic+review%5BTitle%5D)+AND+%22Int+J+Stem+Cells%22%5BJournal%5D)">Methods of isolation and characterization of stem cells from different regions of oral cavity using markers: A systematic review.</a> Int J Stem Cells. 10 (1), 12-20 (2017).</li>
<li>Galvao, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rock+inhibition+drives+resolution+of+acute+inflammation+by+enhancing+neutrophil+apoptosis%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">Rock inhibition drives resolution of acute inflammation by enhancing neutrophil apoptosis.</a> Cells. 8 (9), 964(2019).</li>
</ol>

The authors declare that they have no conflicts of interest.

This protocol describes how to isolate and enrich the BAMBIhighMFGE8high 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 BAMBIhighMFGE8high cells.
First, the most critical technical aspect to consider is the use of an appropriate cell dissociation solution in the present protocol. Although conve...

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 diseases3,4,5. 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 functions7,8 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 BAMBIhighMFGE8high 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 BAMBIhighMFGE8high MSCs in other disorders to understand their authentic functions.
This protocol describes methods for isolating the BAMBIhighMFGE8high subgroup from human UC-MSCs via fluorescence-activated cell sorting (FACS) by flow cytometry and the characteristics of the BAMBIhighMFGE8high subgroup.

<table><tbody><tr><td>0.6 mL microcentrifuge tube</td><td>Corning&amp;nbsp;</td><td>Axygen MCT-060-A</td><td></td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td>Beijing Labgic Technology</td><td>MCT-001-150</td><td></td></tr><tr><td>100 mm cell culture dish&amp;nbsp;</td><td>Beijing Labgic Technology</td><td>12311</td><td></td></tr><tr><td>12 well plate</td><td>Beijing Labgic Technology</td><td>11210</td><td></td></tr><tr><td>15 mL centrifuge tube</td><td>Nanjing Vazyme Material Technology</td><td>TCF00115</td><td></td></tr><tr><td>24 well plate</td><td>Beijing Labgic Technology</td><td>11310</td><td></td></tr><tr><td>50 mL centrifuge tube</td><td>Nanjing Vazyme Material Technology</td><td>TCF00150</td><td></td></tr><tr><td>5 mL Round-Bottom Tubes</td><td>Corning</td><td>FALCON 352003</td><td></td></tr><tr><td>70 &amp;mu;m cell strainer</td><td>Falcon</td><td>352350</td><td>Dilution: 1:1000</td></tr><tr><td>APC anti-human CD79a (Ig&amp;alpha;) Antibody</td><td>BioLegend</td><td>333505</td><td>581&lt;br/&gt; Dilution: 1:200</td></tr><tr><td>APC/Cyanine7 anti-human CD73 (Ecto-5&#039;-nucleotidase) Antibody</td><td>BioLegend</td><td>344022</td><td>G46-6&lt;br/&gt; Dilution: 1:200</td></tr><tr><td>APC-Cy7 Mouse IgG1, &amp;kappa; Isotype Control</td><td>BD Bioscience</td><td>557873</td><td>MOPC-31C (Isotype Control)&lt;br/&gt; Dilution: 1:200</td></tr><tr><td>BAMBI antibody</td><td>Bioss</td><td>bs-12418R</td><td></td></tr><tr><td>Brilliant Violet 510 anti-mouse/human CD44 Antibody</td><td>BioLegend</td><td>103044</td><td>5E10&lt;br/&gt; Dilution: 1:200</td></tr><tr><td>Brilliant Violet 510 Rat IgG2b, &amp;kappa; Isotype Ctrl Antibody</td><td>BioLegend</td><td>400646</td><td>MOPC-21 (Isotype Control)&lt;br/&gt; Dilution: 1:200</td></tr><tr><td>CD105 (Endoglin) Monoclonal Antibody&amp;nbsp; APC</td><td>eBioscience</td><td>17-1057-42</td><td>HM47&lt;br/&gt; Dilution: 1:200</td></tr><tr><td>Cell Counting Chamber Slides</td><td>Shanghai QIUJING</td><td>XB-K-25</td><td></td></tr><tr><td>Centrifuge</td><td>Beijing BAIYANG</td><td>BY-320C</td><td></td></tr><tr><td>ChamQ Universal SYBR qPCR Master Mix</td><td>Vazyme</td><td>Q711-02</td><td></td></tr><tr><td>Chloroform</td><td>XILONG Scientific</td><td>13700908</td><td></td></tr><tr><td>DMEM/F-12 (1:1) basic (1x)</td><td>Gibco</td><td>C11330500BT</td><td></td></tr><tr><td>EDTA (0.5 M), pH 8.0, Rnase free</td><td>Invitrogen</td><td>AM9260G</td><td>Dilution: 1:1000</td></tr><tr><td>Ethanol&amp;nbsp;&amp;nbsp;</td><td>XILONG Scientific</td><td>12803405</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>10099-141C&amp;nbsp;</td><td></td></tr><tr><td>FITC Mouse Anti-Human CD34</td><td>BD Bioscience</td><td>555821</td><td>IM7&lt;br/&gt; Dilution: 1:200</td></tr><tr><td>FITC Mouse Anti-Human CD45</td><td>BD Bioscience</td><td>555482</td><td>AD2&lt;br/&gt; Dilution: 1:200</td></tr><tr><td>FITC Mouse Anti-Human HLA-DR</td><td>BD Bioscience</td><td>555811</td><td>SN6&lt;br/&gt; Dilution: 1:200</td></tr><tr><td>Flow Cytometer</td><td>BD Bioscience</td><td>FACSAria&amp;trade; III Cell SorterAria</td><td></td></tr><tr><td>Flowjo</td><td>BD Bioscience</td><td>V10</td><td></td></tr><tr><td>Gentle Cell Dissociation Reagent</td><td>STEMCELL Technologies</td><td>100-0485</td><td></td></tr><tr><td>Goat Anti-Mouse IgG H&amp;amp;L (Alexa Fluor 488)</td><td>abcam</td><td>ab150113</td><td>HI30&lt;br/&gt; Dilution: 1:200</td></tr><tr><td>Goat Anti-Rabbit IgG H&amp;amp;L (AlexaFluor 594)</td><td>abcam</td><td>ab150080</td><td>Dilution: 1:1000</td></tr><tr><td>HiScript II Q RT SuperMix for qPCR (+gDNA wiper)</td><td>Vazyme</td><td>R223-01</td><td></td></tr><tr><td>Inverted Microscopes</td><td>Nikon</td><td>ECLIPSE Ts2</td><td></td></tr><tr><td>Isopropyl Alcohol</td><td>XILONG Scientific</td><td>12802505</td><td></td></tr><tr><td>MFGE8 antibody</td><td>Biorbyt</td><td>orb388429</td><td>Dilution: 1:100</td></tr><tr><td>Microcentrifuge</td><td>Thermo Fisher Scientific</td><td>FRESCO 21</td><td></td></tr><tr><td>Mouse IgG1 kappa Isotype Control APC</td><td>eBioscience</td><td>17-4714-42</td><td>P3.6.2.8.1 (Isotype Control)&lt;br/&gt; Dilution: 1:200</td></tr><tr><td>Mouse IgG1 kappa Isotype Control FITC</td><td>eBioscience</td><td>11-4714-42</td><td>eBMG2b (Isotype Control)&lt;br/&gt; Dilution: 1:200</td></tr><tr><td>Mouse IgG2b kappa Isotype Control FITC</td><td>eBioscience</td><td>11-4732-42</td><td>RTK4530 (Isotype Control)&lt;br/&gt; Dilution: 1:200</td></tr><tr><td>PBS (10x)</td><td>Sangon Biotech (Shanghai)</td><td>E607016-0500</td><td></td></tr><tr><td>PE-Cy5 Mouse Anti-Human CD90&amp;nbsp;</td><td>BD Bioscience</td><td>555597</td><td>P3.6.2.8.1 (Isotype Control)&lt;br/&gt; Dilution: 1:200</td></tr><tr><td>PE-Cy5 Mouse IgG1 &amp;kappa; Isotype Control</td><td>BD Bioscience</td><td>550618</td><td></td></tr><tr><td>Penicillin-Streptomycin 100x</td><td>Cytiva</td><td>SV30010</td><td>Dilution: 1:100</td></tr><tr><td>Real-Time PCR System</td><td>Applied Biosystems byThermo Fisher Scientific</td><td>Q6</td><td></td></tr><tr><td>RNase-free water</td><td>QIAGEN</td><td>129112</td><td></td></tr><tr><td>Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>NanoDrop One(840-317400)</td><td></td></tr><tr><td>Sterile micropipette tips</td><td>Beijing Labgic Technology</td><td></td><td>Dilution: 1:100</td></tr><tr><td>T75 cell culture flask</td><td>Beijing Labgic Technology</td><td>13212A</td><td></td></tr><tr><td>Thermal Cycler</td><td>Applied Biosystems byThermo Fisher Scientific</td><td>Veriti</td><td></td></tr><tr><td>Tri reagent</td><td>Sigma Aldrich</td><td>T9424</td><td></td></tr><tr><td>Typsin-EDTA Solution</td><td>Bio-Channel Biotechnology</td><td>BC-CE-005</td><td></td></tr><tr><td>Water-Jacketed CO&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp; Incubator</td><td>Thermo Fisher Scientific</td><td>3111</td><td></td></tr></tbody></table>

isolation of human bambihighmfge8high umbilical cord-derived mesenchymal stromal cells

Isolation of Human BAMBIhighMFGE8high Umbilical Cord-Derived Mesenchymal Stromal Cells

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, BAMBIhighMFGE8high 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 BAMBIhighMFGE8high 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 BAMBIhighMFGE8high 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 BAMBIhighMFGE8high UC-MSCs are identified by RT-qPCR. This protocol results in highly efficient and pure cell sorting and describes the marker profiles of the BAMBIhighMFGE8high UC-MSCs.

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 BAMBIhighMFGE8high 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 of BAM...

The isolation of BAMBIhighMFGE8high 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 BAMBIhighMFGE8high UC-MSCs.

Umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) present low immunogenicity and potent immunomodulatory effects for treating various ...

isolation-human-bambihighmfge8high-umbilical-cord-derived-mesenchymal

Research

JoVE Journal

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Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton's jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

Human umbilical cord (UC) can be obtained during the perinatal period as a result of preterm, term, and postterm delivery. In this protocol, we describe the isolation and characterization of UC-derived mesenchymal stem cells (UC-MSCs) from fetuses/infants at 19-40 weeks of gestation.

Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants

This study presents a unique blunt dissection procedure to preserve the integrity of Wharton's jelly (WJ), resulting in less damaged WJ and a greater quantity and viability of the harvested mesenchymal stem cells (MSCs). The method demonstrates superior yield and proliferative ability compared to conventional sharp dissection methods.

Isolation of Umbilical Cord-Derived Mesenchymal Stem Cells with High Yields and Low Damage

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Learn to isolate BAMBI high MFGE8 high human UC-MSCs using flow cytometry and single-cell transcriptomic analysis. This method reveals distinct phenotypes and supports selecting optimal subgroups for treating lupus nephritis.

Isolation of Human BAMBI^highMFGE8^high Umbilical Cord-Derived Mesenchymal Stromal Cells

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