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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+multipotent+cells+in+adult+human+mesenchymal+cell+populations.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wharton's+jelly-derived+cells+are+a+primitive+stromal+cell+population.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+stem+cells+in+human+umbilical+cord+stroma:+In+situ+and+in+vitro+surveys.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+mesenchymal+stem+cells+from+the+sub-amniotic+human+umbilical+cord+lining+membrane.">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/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. L., 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=Isolation+and+characterization+of+human+umbilical+cord+mesenchymal+stem+cells+with+hematopoiesis-supportive+function+and+other+potentials.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+to+isolate+mesenchymal-like+cells+from+Wharton's+Jelly+of+umbilical+cord.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+optimal+culture+conditions+for+clinical+scale+production+of+human+mesenchymal+stem+cells.">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/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>

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 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 ...

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Developmental Biology

5.3K Views.  NCR Biotech Science Cluster. We describe a protocol for the isolation of mesenchymal stem cells from human umbilical cord tissue and their differentiation into the skeletal muscle lineage.

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

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

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 bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

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.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

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

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;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.

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies have demonstrated the wide differentiation capacity that these cells possess. The ovary represents a promising candidate for cell-based therapies due to the fact that it is rich in MSCs and that it is frequently discarded after ovariectomy surgeries as biological waste. This article describes procedures for the isolation, expansion, and differentiation of MSCs derived from the canine ovary, without the necessity of cell-sorting techniques. This protocol represents an important tool for regenerative medicine because of the broad applicability of these highly differentiable cells in clinical trials and therapeutic uses.

Herein, we describe a method for the isolation, expansion, and differentiation of mesenchymal stem cells from canine ovarian tissue.

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...

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

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally isolated and characterized from bone marrow (BM), then acquired from tissues including adipose tissue, synovium, skin, dental pulp, and fetal appendages such as placenta, umbilical cord blood (UCB), and umbilical cord (UC). MSCs are a heterogeneous cell population with the capacity for (1) adherence to plastic in standard culture conditions, (2) surface marker expression of CD73+/CD90+/CD105+/CD45-/CD34-/CD14-/CD19-/HLA-DR- phenotypes, and (3) trilineage differentiation into adipocytes, osteocytes, and chondrocytes, as currently defined by the International Society for Cellular Therapy (ISCT). Although BM is the most widely used source of MSCs, the invasive nature of BM aspiration ethically limits its accessibility. Proliferation and differentiation capacity of MSCs obtained from BM generally decline with the age of the donor. In contrast, fetal MSCs obtained from UC have advantages such as vigorous proliferation and differentiation capacity. There is no ethical concern for UC sampling, as it is typically regarded as medical waste. Human UC starts to develop with continuing growth of the amniotic cavity at 4-8 weeks of gestation and keeps growing until reaching 50-60 cm in length, and it can be isolated during the whole newborn delivery period. To gain insight into the pathophysiology of intractable diseases, we have used UC-derived MSCs (UC-MSCs) from infants delivered at various gestational ages. In this protocol, we describe the isolation and characterization of UC-MSCs from fetuses/infants at 19-40 weeks of gestation.

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.