Name: Generation of Mesenchymal Stem Cells from Human Umbilical Cord Tissue and their Differentiation into the Skeletal Muscle Lineage (Video) | JoVE Journal
Uploaded: 2022-08-31T13:00:00.000+00:00
Duration: 7 min 27 s
Description: Learn to isolate mesenchymal stem cells from human umbilical cord tissue and differentiate them into skeletal muscle lineage. This protocol ensures high viability with minimal endothelial contamination for regenerative medicine research.

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5 minutes

We thank Mr. Ojas Tikoo for their help with filming and video production. We also acknowledge the help received from the GARBH-Ini (Interdisciplinary Group on Advanced Research and Birth Outcome-DBT India) staff, nurses, and senior research officers at the Gurugram Civil Hospital and Dr. Pallavi Kshetrapal for help with logistics. This work was supported by grants awarded to Suchitra Gopinath from the Department of Biotechnology, India (BT/09/IYBA/2015; BT/PR29599/PFN/20/1393/2018).

The use of umbilical cord tissue in this study was approved by the Institutional Committee for Stem Cell Research (IC-SCR), the Institutional Ethics Committee, Translational Health Science and Technology Institute (IEC-THSTI), the Institutional Ethics Committee of Civil Hospital, Gurugram, Haryana, and the Institutional Biosafety Committee, THSTI. Human cord tissue samples were harvested from term deliveries at the time of birth. Informed written consent was obtained from subjects . All methods were carried out in accordance with relevant guidelines and regulations.
1. Isolation of MSCs from cord tissue
<ol>
	<li>At the time of delivery, cut at least 5 cm of cord, preferably closer to the placenta, and sterilize the cord by swabbing the outer surface with 70% ethanol. Transfer the piece of cord tissue sequentially from one 50 mL collection tube containing phosphate-buffered saline (PBS) to another containing the same. Transport the collection tube bearing the name of the subject on ice to the laboratory within 1 h. 
	NOTE: For a larger study, it might be useful to barcode the samples to enable their tracking throughout the study. Importantly, all personnel handling human tissues should be offered the full regimen of the hepatitis B vaccine.</li>
	<li>In the lab, switch on the UV for 25 min in the BSL-2 hood to sterilize autoclaved instruments and pipettes prior to use.</li>
	<li>Transfer the cord tissue piece from the collection tube to a 10 cm2 tissue culture-treated dish containing PBS enriched with 5 g/L glucose, 50 &#181;g/mL gentamicin, 2.5 &#181;g/mL amphotericin B, 100 U/mL penicillin, and 100 &#181;g/mL streptomycin (schematic in Figure 1).</li>
	<li>Using a scalpel, slice the cord tissue vertically along its longitudinal axis to obtain two half cylindrical pieces. Owing to cord torsion and the mucoid surface, pin down the tissue with a pair of forceps held in the other hand.</li>
	<li>At this point, observe the umbilical arteries and vein and remove the blood vessels by using a scalpel to scrape them off in one direction from the surface. Rinse the cord tissue once again in PBS to remove all residual blood associated with the tissue. Ensure that the scraping is gentle to preserve the integrity of cells in the WJ surrounding the vessels.</li>
	<li>Mince each half of the cord tissue into 0.5 cm3 sized fragments and place the fragments with the luminal surface facing down on the dish. Incubate the dish briefly for 10 min in a 37 &#176;C humidified chamber containing 5% CO2.</li>
	<li>After incubation, flood the dish containing the cord tissue pieces with 20 mL of medium containing MEM Alpha Modification without L-glutamine, ribo- and deoxyribonucleosides, 15% fetal bovine serum (not heat-inactivated), and 50 &#181;g/mL gentamycin. Add the growth medium gently along the sides to prevent the tissue explants from being dislodged from their orientation. Add excess medium to account for a fraction that will be soaked up by the tissue explants during incubation.</li>
	<li>After 3 days of incubation, add fresh medium to the cultures. Ensure that the cultures are protected from shocks and movement of the explants while handling the dishes.</li>
	<li>After 1 week, remove the tissue fragments individually using sterile forceps and discard using appropriate biohazard bags for disposal. Retain the existing medium and add 10 mL of fresh growth medium. Replace the growth medium every 4 days until individual colonies reach a confluence of 70%. 
	NOTE: It is likely that the cells will not be uniformly distributed throughout the dish, as there will be individual proliferative colonies that need to be monitored for confluence with time. Generally, within a month, a 10 cm2 dish generates enough cells to be split into a separate dish.</li>
	<li>Harvest the adherent cells using trypsin/EDTA solution (1x 0.25% trypsin and 0.02% EDTA in Hanks Balanced Salt Solution [HBSS]). Centrifuge the cell suspension at 470 &#215; g for 5 min at 25 &#176;C and resuspend the cell pellet in growth medium.</li>
</ol>
2. Immunophenotyping and propagation of uMSCS
<ol>
	<li>Proceed to immunophenotyping once the adherent cells have reached 50%-60% confluence and are well spread. Do not perform MSC marker analysis on fully confluent cultures, as this tends to cause downregulation of key MSC markers.</li>
	<li>After trypsinization, distribute a cell suspension of 1 &#215; 106 cells/mL in FACS tubes (1 &#215; 105 cells/tube) and stain with appropriate fluorophore-linked antibodies (all 1:50 dilution) in combination: unstained; CD105 + CD90; CD105 + CD73; CD105; CD90; CD73; CD34 + CD45 (common fluorophore); isotype controls for each fluorophore. Record a total number of events of at least 10,000 on the flow cytometer for further analysis. 
	NOTE: Since the cells are analyzed for each surface marker separately, gating on cell subsets is not required.</li>
	<li>In addition to the above markers, confirm the presence of positive and negative surface markers in uMSC lines created from individual cord tissue samples (Table 1).</li>
	<li>Analyze the labeled cells by flow cytometry and determine the percentage of CD105+CD90+ and CD105+CD73+ cells. Analyze CD105+ and CD34&#8722;CD45&#8722; cells separately.</li>
</ol>
3. Differentiation of uMSCs into skeletal muscle
<ol>
	<li>Coat tissue culture plates with 0.01% collagen and 20 &#181;g/mL laminin in PBS. Coat these plates for a minimum of 4 h at room temperature.</li>
	<li>Plate uMSCs at a density of 10,000 cells/cm2 in growth medium.</li>
	<li>When the cells are 70% confluent, aspirate the growth medium and rinse the cultures 2x with PBS. Add myogenic differentiation medium (M1) comprising of DMEM + 5% horse serum + 0.1 &#181;M dexamethasone + 50 &#181;M hydrocortisone to the uMSCs. For determination of the kinetics of myogenic progression, add M1 medium every other day to the cultures.</li>
	<li>To determine the kinetics of myogenic progression, analyze uMSCs for Pax7, MyoD, Myogenin, and MyHC expression at 2 days, 4-5 days, 6-7 days, and 10-14 days, respectively.</li>
</ol>

<ol><li>Kuroda, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unique+multipotent+cells+in+adult+human+mesenchymal+cell+populations%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Unique multipotent cells in adult human mesenchymal cell populations.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (19), 8639-8643 (2010).</li>
<li>Troyer, D. L., Weiss, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wharton%27s+jelly-derived+cells+are+a+primitive+stromal+cell+population%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Wharton's jelly-derived cells are a primitive stromal cell population.</a> Stem Cells. 26 (3), 591-599 (2008).</li>
<li>Karahuseyinoglu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biology+of+stem+cells+in+human+umbilical+cord+stroma%3A+In+situ+and+in+vitro+surveys%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Biology of stem cells in human umbilical cord stroma: In situ and in vitro surveys.</a> Stem Cells. 25 (2), 319-331 (2007).</li>
<li>Kita, K., Gauglitz, G. G., Phan, T. T., Herndon, D. N., Jeschke, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+characterization+of+mesenchymal+stem+cells+from+the+sub-amniotic+human+umbilical+cord+lining+membrane%5BTitle%5D)+AND+%22Stem+Cells+and+Development%22%5BJournal%5D)">Isolation and characterization of mesenchymal stem cells from the sub-amniotic human umbilical cord lining membrane.</a> Stem Cells and Development. 19 (4), 491-502 (2010).</li>
<li>Sarugaser, R., Lickorish, D., Baksh, D., Hosseini, M. M., Davies, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+umbilical+cord+perivascular+%28HUCPV%29+cells%3A+A+source+of+mesenchymal+progenitors%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Human umbilical cord perivascular (HUCPV) cells: A source of mesenchymal progenitors.</a> Stem Cells. 23 (2), 220-229 (2005).</li>
<li>Mishra, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Umbilical+cord+tissue+is+a+robust+source+for+mesenchymal+stem+cells+with+enhanced+myogenic+differentiation+potential+compared+to+cord+blood%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Umbilical cord tissue is a robust source for mesenchymal stem cells with enhanced myogenic differentiation potential compared to cord blood.</a> Scientific Reports. 10 (1), 18978(2020).</li>
<li>Lu, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+characterization+of+human+umbilical+cord+mesenchymal+stem+cells+with+hematopoiesis-supportive+function+and+other+potentials%5BTitle%5D)+AND+%22Haematologica%22%5BJournal%5D)">Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials.</a> Haematologica. 91 (8), 1017-1026 (2006).</li>
<li>Seshareddy, K., Troyer, D., Weiss, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Method+to+isolate+mesenchymal-like+cells+from+Wharton%27s+Jelly+of+umbilical+cord%5BTitle%5D)+AND+%22Methods+in+Cell+Biology%22%5BJournal%5D)">Method to isolate mesenchymal-like cells from Wharton's Jelly of umbilical cord.</a> Methods in Cell Biology. 86, 101-119 (2008).</li>
<li>Sotiropoulou, P. A., Perez, S. A., Salagianni, M., Baxevanis, C. N., Papamichail, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+the+optimal+culture+conditions+for+clinical+scale+production+of+human+mesenchymal+stem+cells%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells.</a> Stem Cells. 24 (2), 462-471 (2006).</li>
<li>Yoon, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+explant-derived+and+enzymatic+digestion-derived+MSCs+and+the+growth+factors+from+Wharton%27s+jelly%5BTitle%5D)+AND+%22BioMed+Research+International%22%5BJournal%5D)">Comparison of explant-derived and enzymatic digestion-derived MSCs and the growth factors from Wharton's jelly.</a> BioMed Research International. 2013, 428726(2013).</li>
<li>Ishige, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+mesenchymal+stem+cells+derived+from+arterial%2C+venous%2C+and+Wharton%27s+jelly+explants+of+human+umbilical+cord%5BTitle%5D)+AND+%22International+Journal+of+Hematology%22%5BJournal%5D)">Comparison of mesenchymal stem cells derived from arterial, venous, and Wharton's jelly explants of human umbilical cord.</a> International Journal of Hematology. 90 (2), 261-269 (2009).</li>
<li>Chal, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differentiation+of+pluripotent+stem+cells+to+muscle+fiber+to+model+Duchenne+muscular+dystrophy%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy.</a> Nature Biotechnology. 33 (9), 962-969 (2015).</li>
</ol>

The authors declare no competing interests.

Critical steps 
A critical step in this protocol is the collection of tissue under aseptic conditions, from the time of delivery to the maintenance of sterile cultures, for the entire duration of propagation. During cord collection, it is essential that the cord does not touch any non-sterilized surface and is externally swabbed with 70% ethanol before collection in tubes containing PBS supplemented with antibiotics. It is important to limit the time between cord collection and processing of the tis...

The human umbilical cord has been credited to possess a robust reservoir of mesenchymal stem cells, which are currently being explored for regenerative therapies due to their robust proliferation and differentiation rates, immunomodulatory properties, and ability to generate cells from all the three germ layers1. The umbilical cord tissue consists of multiple compartments such as the umbilical cord blood, the umbilical vein subendothelium, and the Wharton's jelly (WJ), which in itself encompasses three indistinct regions-the perivascular zone, the intervascular zone, and the sub-amnion or the cord lining (CL)2. While uMSCs can be isolated from all these different regions and broadly express key MSC markers, there is no clarity on whether these compartments contain the same population of uMSCs or display differences in their differentiation potencies3. Hence, protocols for the isolation of uMSCs require a greater precision in their mode and region of isolation, the robust characterization of differentiation potentials, and finally, a comparative analysis from different compartments of the cord. 
In this context, few studies have demonstrated differences in uMSC proliferative and differentiative potentials between different parts of the cord. Of these, comparative analyses between uMSCs isolated from the CL and WJ regions revealed a greater proliferative potential in CL-derived uMSCs3,4. In a separate study, WJ-derived uMSCs performed better in proliferation assays compared to perivascular cells (HUCPV)5. In examining differences between cord blood-derived uMSCs and cord tissue-derived uMSCs devoid of vascular contamination, differential expression of key MSC markers was reported between the two compartments, as well as increased proliferation rates in cord tissue-derived uMSCs6. 
Of the several studies examining the differentiation potentials of uMSCs primarily into tissues of the mesoderm lineage such as osteogenic, adipogenic, and chondrogenic lineages, very few have provided detailed protocols for myogenic differentiation and subsequent characterization, as well as comparative analyses between various cord compartments. In this context, we have developed a robust muscle differentiation protocol and observed that cord tissue-derived uMSCs display superior myogenic differentiation capabilities compared to cord blood6. Here, a stepwise protocol is detailed for the isolation of uMSCs from the whole cord tissue devoid of cells associated with the vasculature, their characterization, and their differentiation into the myogenic lineage.

<table><tbody><tr><td>4&#039;,6-diamidino-2-phenylindole (DAPI)</td><td>Thermo Fisher Scientific</td><td>D1306</td><td></td></tr><tr><td>Amphotericin B</td><td>Sigma Aldrich</td><td>A2411</td><td></td></tr><tr><td>Antibiotic solution 100x Liquid, endotoxin tested (10,000 U Penicillin and 10 mg Streptomycin/mL in 0.9% normal saline)</td><td>HiMedia</td><td>A001A-50mL</td><td></td></tr><tr><td>Anti-GAPDH antibody</td><td>Sigma Aldrich</td><td>G8795</td><td></td></tr><tr><td>Anti-MyHC antibody (My32)</td><td>Novus Biologicals</td><td>NBP2-50401AF647</td><td></td></tr><tr><td>Anti-MyoD antibody (5.8A)</td><td>Novus Biologicals</td><td>NB100-56511</td><td></td></tr><tr><td>Anti-Myogenin antibody (Clone F5D)</td><td>Novus Biologicals</td><td>NBP2-34616AF594</td><td></td></tr><tr><td>Anti-Pax7 antibody</td><td>DSHB</td><td>DSHB-C1-576</td><td></td></tr><tr><td>APC Mouse anti-human CD90 clone 5E10</td><td>BD Biosciences</td><td>559869</td><td></td></tr><tr><td>Collagen Type 1</td><td>Merck</td><td>C8919</td><td></td></tr><tr><td>D (+) Glucose</td><td>Sigma Aldrich</td><td>G7021</td><td></td></tr><tr><td>Dexamethasone</td><td>SIGMA</td><td>D4902</td><td></td></tr><tr><td>FACSCanto II or FACSAria III</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Fetal Bovine Serum, qualified Brazil</td><td>GIBCO</td><td>10270106</td><td>not to be heat-inactivated</td></tr><tr><td>FITC Mouse anti-human CD106 clone 51-10C9</td><td>BD Biosciences</td><td>551146</td><td></td></tr><tr><td>FITC Mouse anti-human CD14 clone M5E2</td><td>BD Biosciences</td><td>557153</td><td></td></tr><tr><td>FITC Mouse anti-human CD31 clone WM59</td><td>BD Biosciences</td><td>557508</td><td></td></tr><tr><td>FITC Mouse anti-human CD34 clone 581</td><td>BD Biosciences</td><td>555821</td><td></td></tr><tr><td>FITC Mouse anti-human CD45 clone HI30</td><td>BD Biosciences</td><td>555482</td><td></td></tr><tr><td>FITC Mouse anti-human CD49D clone 9F10</td><td>BD Biosciences</td><td>560840</td><td></td></tr><tr><td>FITC Mouse anti-human CD90 clone 5E10</td><td>BD Biosciences</td><td>555595</td><td></td></tr><tr><td>FITC Mouse anti-human HLA-A,B,C clone G46-2.6</td><td>BD Biosciences</td><td>557348</td><td></td></tr><tr><td>FITC Mouse anti-human IgG clone G18-145</td><td>BD Biosciences</td><td>555786</td><td></td></tr><tr><td>FlowJo software</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Gentamicin</td><td>Sigma Aldrich</td><td>G1264</td><td></td></tr><tr><td>Horse serum</td><td>HiMedia</td><td>RM1239</td><td></td></tr><tr><td>Hydrocortisone</td><td>Merck</td><td>H4001</td><td></td></tr><tr><td>Laminin</td><td>Merck</td><td>L2020</td><td></td></tr><tr><td>MEM Alpha Modification without L-glutamine, ribo- and deoxyribonucleosides</td><td>Hyclone</td><td>SH30568.FS</td><td>Basal medium for uMSCs</td></tr><tr><td>PE Mouse anti-human CD105 clone 266</td><td>BD Biosciences</td><td>560839</td><td></td></tr><tr><td>PE Mouse anti-human CD44 clone 515</td><td>BD Biosciences</td><td>550989</td><td></td></tr><tr><td>PE Mouse anti-human CD49E clone llA1</td><td>BD Biosciences</td><td>555617</td><td></td></tr><tr><td>PE Mouse anti-human IgG clone G18-145</td><td>BD Biosciences</td><td>555787</td><td></td></tr><tr><td>PE-Cy7 Mouse anti-human CD73 CLONE AD2</td><td>BD Biosciences</td><td>561258</td><td></td></tr><tr><td>Phosphate buffered saline (PBS), pH=7.4</td><td>HiMedia</td><td>M1866</td><td></td></tr><tr><td>Trypsin/EDTA solution (1x 0.25% Trypsin and 0.02% EDTA in Hanks Balanced Salt Solution (HBSS)</td><td>HiMedia</td><td>TCL049-100mL</td><td></td></tr></tbody></table>

generation of mesenchymal stem cells from human umbilical cord tissue and their differentiation into the skeletal muscle lineage

Generation of Mesenchymal Stem Cells from Human Umbilical Cord Tissue and their Differentiation into the Skeletal Muscle Lineage

Exploring the therapeutic potential of mesenchymal stem cells is contingent upon the ease of isolation, potency toward differentiation, and the reliability and robustness of the source. We describe here a stepwise protocol for the isolation of mesenchymal stem cells from human umbilical cord tissue (uMSCs), their immunophenotyping, and the propagation of such cultures over several passages. In this procedure, the viability of the uMSCs is high because there is no enzymatic digestion. Further, the removal of blood vessels, including the umbilical cord arteries and the vein, ensures that there is no contamination of cells of endothelial origin. Using flow cytometry, uMSCs upon isolation are CD45&#8722;CD34&#8722;, indicating an absence of cells from the hematopoietic lineage. Importantly, they express key surface markers, CD105, CD90, and CD73. Upon establishment of cultures, this paper describes an efficient method to induce differentiation in these uMSCs into the skeletal muscle lineage. A detailed analysis of myogenic progression in differentiated uMSCs reveals that uMSCs express Pax7, a marker for myogenic progenitors in the initial stages of differentiation, followed by the expression of MyoD and Myf5, and, finally, a terminal differentiation marker, myosin heavy chain (MyHC).

The success of isolation of uMSCs from cord tissue is &gt;95%, unlike the poor rates of success from whole cord blood. Upon successful isolation of uMSCs, FACS analysis reveals that all the cells are CD34−CD45−CD105+CD90+. However, in comparative analysis, uMSCs isolated from cord blood display heterogeneous populations, wherein a proportion of cells show CD34+CD45+CD105+ (~15%). Additionally, double-positive CD105+CD90+ ar...

We describe a protocol for the isolation of mesenchymal stem cells from human umbilical cord tissue and their differentiation into the skeletal muscle lineage.

Exploring the therapeutic potential of mesenchymal stem cells is contingent upon the ease of isolation, potency toward differentiation, and the ...

generation-mesenchymal-stem-cells-from-human-umbilical-cord-tissue

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Learn to isolate mesenchymal stem cells from human umbilical cord tissue and differentiate them into skeletal muscle lineage. This protocol ensures high viability with minimal endothelial contamination for regenerative medicine research.

Generation of Mesenchymal Stem Cells from Human Umbilical Cord Tissue and their Differentiation into the Skeletal Muscle Lineage (Video) | JoVE Journal

Generation of Mesenchymal Stem Cells from Human Umbilical Cord Tissue and their Differentiation into the Skeletal Muscle Lineage

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