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

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 ...

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

Research

JoVE Journal

Developmental Biology

5.5K Views.  NCR Biotech Science Cluster. We beschrijven ons protocol om mesenchymale stamcellen te isoleren van menselijk navelstrengweefsel en een robuuste methode om ze te differentiëren in skeletspierlijn. Deze techniek stelt ons in staat om mesenchymale stamcellen te isoleren die een hoge levensvatbaarheid en warmte bezitten, met weinig tot geen besmetting van cellen van endotheeloorsprong en om myogene differentiatie efficiënt te induceren bij deze ziekten. Koordtorsie en mucoid consistentie maken het moeilijk om het snoer te...

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

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Bioengineering

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 ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

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.

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

Isolating Stem Cells from Soft Musculoskeletal Tissues

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of excitement for treating injured and diseased tissues due to their impressive capabilities to undergo multi-lineage cell differentiation and their self-renewal ability. Most importantly, these qualities have made them advantageous for use in autologous cell transplantation therapies. The current protocol will introduce the readers to the modified preplate technique where soft tissues of the musculoskeletal system, e.g. tendon and muscle, are 1st enzymatically dissociated and then placed in collagen coated flasks with medium. The supernatant, which is composed of medium and the remaining floating cells, is serially transferred daily to new flasks. The stem cells are the slowest to adhere to the flasks which is usually takes 5-7 days (serial transfers or preplates) . By using this technique, adult stem cells present in these tissues can be easily harvested through fairly non-invasive procedures.

Isolating adult stem cells from musculoskeletal soft tissues based on the cell's adherence speed to flask.

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

Biology

Isolation and Culture of Three Kinds of Umbilical Cord Mesenchymal Stem Cells

Umbilical cord mesenchymal stem cells (UC-MSCs) are an important cell source for regenerative medicine. UC-MSCs can be isolated from the umbilical cord Wharton's jelly, as well as from the umbilical arteries and umbilical vein. They are known as perivascular stem cells obtained from umbilical arteries (UCA-PSCs), perivascular stem cells obtained from the umbilical vein (UCV-PSCs), and mesenchymal stem cells obtained from Wharton's jelly (WJ-MSCs). UCA-PSCs and UCV-PSCs are pericytes derived from perivascular regions that are progenitors of MSCs. Isolation and culture of the three kinds of cells is an important source for studying stem cell transplantation and repair. The present protocol focuses on the isolation and culture of cells through mechanical separation, adherent culture, and cell crawling out. Through this technique, the three different types of stem cells can be derived. Cell surface markers were detected by flow cytometry. The stem cells were detected for multilineage differentiation potential by adipogenic, osteogenic, and neural-like differentiation, which is consistent with the phenotype of MSCs. This experimental protocol expands the source of UC-MSCs. In addition, the cell isolation method provides a basis for further study of regenerative medicine and other applications.

The present protocol describes the isolation and culture of mesenchymal stem cells from the umbilical cord arteries, vein, and Wharton's jelly.

Umbilical cord mesenchymal stem cells (UC-MSCs) are an important cell source for regenerative medicine. UC-MSCs can be isolated from the umbilical cord ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

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

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