Name: generation of mesenchymal stem cells from human umbilical cord tissue and their differentiation into the skeletal muscle lineage
Uploaded: 2022-08-31T13:00:00
Description: Watch this Scientific Journal Video about Generation of Mesenchymal Stem Cells from Human Umbilical Cord Tissue and their Differentiation into the Skeletal Muscle Lineage at JoVE.com

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 accord...

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+umbilical+cord+perivascular+(HUCPV)+cells:+A+source+of+mesenchymal+progenitors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Umbilical+cord+tissue+is+a+robust+source+for+mesenchymal+stem+cells+with+enhanced+myogenic+differentiation+potential+compared+to+cord+blood.">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. 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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+explant-derived+and+enzymatic+digestion-derived+MSCs+and+the+growth+factors+from+Wharton's+jelly.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+mesenchymal+stem+cells+derived+from+arterial,+venous,+and+Wharton's+jelly+explants+of+human+umbilical+cord.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+pluripotent+stem+cells+to+muscle+fiber+to+model+Duchenne+muscular+dystrophy.">Differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy.</a> Nature Biotechnology. 33 (9), 962-969 (2015).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4&#39;,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

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 &#62;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&#8722;CD45&#8722;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<s...

Watch this Scientific Journal Video about Generation of Mesenchymal Stem Cells from Human Umbilical Cord Tissue and their Differentiation into the Skeletal Muscle Lineage at JoVE.com

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

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 ...

We describe our protocol to isolate mesenchymal stem cell from human umbilical cord tissue and a robust method to differentiate them into skeletal muscle lineage. This technique allows us to isolate mesenchymal stem cells possessing high viability and heat, with little to no contamination of cells of endothelial origin and to induce myogenic differentiation efficiently in these illnesses. Cord torsion and mucoid consistency make it difficult to handle the cord.We show how to process the cord, to not scrape off the Wharton's jelly, and exclude endothelial cells from the population. After collecting the cord tissue at the delivery time, transfer the cord tissue piece from the collection tube to a 10 square centimeter tissue culture treated dish, and wash the tissue thoroughly with fresh PBS. To care for the cord torsion and mucoid surface, pin down the tissue with a pair of forceps held in one hand.Using a scalpel, slice the cord tissue vertically along its longitudinal access to obtain two half cylindrical pieces. At this point, observe the umbilical arteries and veins. Using a scalpel, remove the blood vessels by scraping them off in one direction from the surface and rinse the cord tissue in PBS to remove all residual blood associated with the tissue.Mince each half of the cord tissue into 0.5 cubic centimeter sized fragments. Place the fragments with the luminal surface facing down on the dish and incubate the dish briefly for 10 minutes. At the end of the incubation, add 20 milliliters of medium containing MEM Alpha modification gently along the sides of the dish containing the cord tissue.Ensure that the explants are not dislodged from their orientation and add excess medium to account for a fraction that the tissue explants will soak up during incubation. Then place the dish in the incubator for three days. After the end of the incubation, add fresh medium to the culture and ensure that the cultures are protected from shocks and movement of the explants while handling the dishes.After one week, using sterile forceps, remove the tissue fragments individually and discard them using appropriate biohazard bags for disposal. Retain the existing medium and add 10 milliliters of fresh growth medium. Replace the growth medium every four days until individual colonies reach a confluence of 70%After staining the cells as described in the manuscript, analyze the labeled cells by flow cytometry, and determine the percentage of CD105 CD90 positive, and CD105 CD73 positive cells.Analyze CD105 positive and CD34 CD45 negative cells separately. Coat the tissue culture plate with 0.01%collagen and 20 micrograms per milliliters laminin in PBS and place it on the rocker for a minimum of four hours at room temperature. After the incubation, remove the collagen and wash the plate with PBS, then plate uMSCs at 10, 000 cells per square centimeter density in the growth medium.Once the cells attain 70%confluency, aspirate the growth medium, and rinse the culture plate twice with PBS. To determine the kinetics of myogenic progression, add M1 medium every other day to the cultures and analyze uMSCs for the expression of pax7, MyoD, myogenin, and myosin heavy chain at two days, four to five days, six to seven days, and ten to fourteen days respectively. uMSCs display the expression of CD105 and CD90, and do not express hematopoietic markers CD34 and CD45.The uMSCs were also positive for the expression of uMSCs marker CD73, indicating that the uMSCs expressed multiple key markers. uMSCs showed expression of pax7 within the first two days of the addition of M1, followed by MyoD expression within the first four days of M1 addition. Cells express myogenin protein at six days of differentiation, followed by the expression of myosin heavy chain between 10 days and 14 days of the induction of differentiation.970 genes were upregulated in response to the induction of myogenic differentiation as compared to the undifferentiated uMSCs. More myogenic genes were upregulated in cord tissue derived uMSCs compared to cord blood derived uMSCs. Both cord tissue and cord blood showed upregulation of genes related to cytoskeletal proteins, associated with actin binding and sarcomere assembly, transporters associated with contractile function, muscle mass maintenance, calcium signaling, and enzymatic function.Collect the tissue under aseptic condition and maintain sterile culture. The cord should be externally swept with 70%ethanol and processed as fast as possible. Many regenerative therapies require large numbers of cells from early passages.It can also be used as a model to mimic the entire dry environment and reflect postnatal metabolism.

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