This work was financially supported by the National Natural Science Foundation of China (82172107), the Natural Science Foundation of Guangdong Province, China (2021A1515011927, 2021A1515010918, 2020A1515110347), Shenzhen Medical Research Fund (SMRF.D2301015), the Shenzhen Municipal Science and Technology Innovation Committee (JCYJ20210324135014040, JCYJ20220530172807016, JCYJ20230807150908018, JCYJ20230807150915031), and Longgang District Special Fund for Economic and Technological Development (LGKCYLWS2022007).

Samples were obtained from the consenting, healthy donors from the Shenzhen Longgang District Maternity and Child Healthcare Hospital, Guangdong, China. The use of human samples for the study was approved by the Ethics Committee of Shenzhen Hospital, Beijing University of Chinese Medicine (SZLDH2020LSYM-095) and Medical Ethics Committee of Shenzhen Longgang District Maternity and Child Healthcare Hospital (LGFYYXLLS-2020-005). All experiments were conducted according to the approved guidelines. The details of the reagents and the equipment used are listed in the Table of Materials.
1. Collection of human umbilical cord
<ol>
	<li>Obtain human umbilical cord samples from the cooperative hospital under sterile conditions. 
	NOTE: Choose a donor under 35 years of age, preferably a primipara, with no history of hepatitis, sexually transmitted diseases, or genetic disorders. The admission examination results for hepatitis B virus, hepatitis C virus, and cytomegalovirus need to be negative. Choose cesarean section as the mode of delivery.</li>
	<li>Select a straight and intact piece of UC of approximately 10-15 cm in length.</li>
	<li>Close both ends with hemostatic forceps and ligate with surgical knots at the inner end between the forceps right after the delivery of a full-term newborn by cesarean section. (collected UC as shown in Figure 1, step 1). 
	NOTE: The operation of the cesarean section ensures the collection is sterile. Use surgical knots of size 0 or 2-0 suture for better tensile strength.</li>
	<li>Cut the tied umbilical cord between the forceps and knots using surgical scissors (Figure 1, step 1).</li>
	<li>Rinse it with saline solution to remove any contaminating blood clots on the surface and transfer promptly to a sterile 50 mL plastic tube containing 20 mL of sterile phosphate-buffered saline (PBS). 
	NOTE: No antibiotic was used during the transfer.</li>
	<li>Seal the tube. Place it on ice in a polystyrene box and store it at 4 &#176;C before transporting it to the laboratory. 
	NOTE: Dissect the cord within 4 h of collection.</li>
</ol>
2. Isolation of Wharton&#39;s jelly from the umbilical cord
<ol>
	<li>Process the umbilical cord following the steps below:
	<ol>
		<li>Prepare a sterilized tray with surgical instruments, aseptically packaged 90 mm dishes, 1000 &#181;L pipettes, sterile PBS, 75% ethanol solution, and xeno-free human MSC culture medium. 
		NOTE: The toothed forceps and mosquito clamps permit the slithery cord to be dealt with efficiently.</li>
		<li>Surface decontaminate the required items and work zone before placing the items inside the biosafety cabinet. 
		NOTE: Allow the work zone air to purge for a few minutes before commencing work.</li>
		<li>Disinfect the surface of the tube and remove the sample into a Petri dish in the biosafety cabinet. Immerse the sample with the knots in 75% ethanol for 30 s and then rinse it multiple times with sterile PBS in a new Petri dish. 
		NOTE: The cord must be ligated before immersing in 75% ethanol, and the time of immersing should be limited to 30 s to 1 min to fully disinfect the cord without damage.</li>
		<li>Cut the knots off and cut the cord between two knots transversally into short pieces about 2 cm in length with surgical scissors and forceps. 
		NOTE: The ends of the cord with knots should be disposed of. The length of the UC shouldn&#39;t be too long or too short; both conditions may increase the difficulty of the operation.</li>
		<li>Perfuse the vein (larger vessel) with PBS using 1000 &#181;L pipettes until the fluid coming out of the other end of the cord becomes completely transparent19.</li>
	</ol>
	</li>
	<li>Dissect the umbilical cord.
	<ol>
		<li>Transfer one of the already cut pieces to a new Petri dish. 
		NOTE: Two arteries and one vein should be exposed visibly on the cross section8 (Figure 2B).</li>
		<li>Add the appropriate amount of PBS to keep the cord wet throughout the operation.</li>
		<li>Grasp the cord with forceps and pull out the arteries along its major axis with the help of mosquito clamps. 
		NOTE: Try to pull from the other end of the cord if the artery has been torn off, as the artery wall has great elasticity.</li>
		<li>Fix the cord horizontally with the thicker side facing up by inserting one blade of the forceps into the umbilical vein and then clamping tightly, ensuring the forceps clamp on the thicker side.</li>
		<li>Make a linear incision on the layer of amniotic epithelium from one end to the other along the edge of the other blade of the forceps using a scalpel (Figure 3). 
		NOTE: Preserve the integrity of the Wharton&#39;s jelly by cutting as little WJ as possible.</li>
		<li>Begin at one corner of the cutting gap. Lift the corner of the amniotic epithelium with toothed clamps and continue to cut horizontally a little further with corneal scissors along the inner surface of the epithelium.
		<ol>
			<li>Separate the Wharton&#39;s jelly and amniotic epithelium with clamps and gradually expand to the whole circle of one end of UC. Peel off the epithelial layer lengthways (Figure 3). 
			&#8203;NOTE: The Wharton&#39;s jelly is a mucous connective tissue enclosed by the dense amniotic epithelium8. The whole operation should be smooth, but mosquito clamps could be used for blunt dissection if one encounters difficulties during peeling.</li>
		</ol>
		</li>
		<li>Insert both blades of the forceps inside the vein, clamp a portion of WJ, and then flip it inside out to expose the epithelium of the umbilical vein (Figure 3).</li>
		<li>Peel off the epithelium of the vein easily, as the internal tension creates a separation between the epithelium of the vein and the perivascular WJ (Figure 3).</li>
	</ol>
	</li>
	<li>Dissect the UC using the conventional method for comparison20.
	<ol>
		<li>Choose another already cut sample of nearly the same weight as the previous cord and transfer it to a new Petri dish.</li>
		<li>Cut along the umbilical vein longitudinally with scissors and spread the UC to expose the vessels and WJ.</li>
		<li>Remove the umbilical vein and two arteries, and scape the WJ off the endothelial layer with scissors and scalpel.</li>
	</ol>
	</li>
</ol>
3. Isolation and culture of UC-MSCs
<ol>
	<li>Place the collected WJ in a pre-labeled 90 mm Petri dish and cut it into 1-3 mm pieces with scissors and forceps.</li>
	<li>Invert the dish with the cap on the bottom and place it in a 5% CO2 incubator at 37 &#176;C for 30 min to strengthen the attachment between the pieces and the plastic surface.</li>
	<li>Turn the dish over, and gently add an appropriate amount of human MSC culture medium for further culture. 
	NOTE: Use the lowest speed to ensure the pieces are still attached.</li>
	<li>Change the human MSC culture medium every 3 days. Exchange two-thirds of the medium and observe the appearance of outgrowth of fibrous cells every 2 days in the first 7 days, change the full medium, and observe it on a daily basis thereafter. 
	NOTE: Avoid moving the Petri dish for the first 7 days. The distinguished outgrowth of fibroblast-like cells was observed in about 4 days.</li>
	<li>Subculture until confluence reaches 80%. 
	NOTE: This step takes 7-10 days.
	<ol>
		<li>Pre-warm the PBS, 0.25% trypsin, and xeno-free human MSC culture medium to 37 &#176;C in a water bath.</li>
		<li>Rinse with PBS after aspirating the supernatant with pipettes, and detach the cells with pre-heated 0.25% trypsin until the cells can be dislodged by tapping the Petri dish.</li>
		<li>Inactivate the trypsin by mixing pre-heated MSC culture medium at a ratio of 4:1, collect the cell suspension, and centrifuge at 300 x g for 5 min at 4 &#176;C. 
		NOTE: These cells are considered passage 0 (P0).</li>
		<li>Discard the supernatant with a pipette, resuspend the cells in the human MSC culture medium, and inoculate into new Petri dishes at a seeding density of 1x104 cells/cm2 for propagation.</li>
	</ol>
	</li>
	<li>Using a hemocytometer, count the total cell number of the two methods after amplification at first, second, and third passage. Determine the cell morphology under the microscope in the first, third, and fifth passages. 
	NOTE: The morphology of these cells should be similar to the P0 cells.</li>
	<li>Use cells at the fifth passage for flow cytometry and other experiments.</li>
</ol>
4. Expression of cell surface markers by flow cytometry
<ol>
	<li>Detach passage 5 (P5) cells with 0.25% trypsin, wash the cells with cell staining buffer, and centrifuge at 300 x g for 5 min at 4 &#176;C. Resuspend the pellets with cell staining buffer to 100 &#181;L each.</li>
	<li>Label each tube and add the appropriate amounts of antibodies, which were conjugated with Allophycocyanin (APC), fluorescein isothiocyanate (FITC) or phycoerythrin (PE): CD44-APC, CD73-APC, CD90-FIFC, CD105-FIFC, CD11B-PE, CD19-PE, CD43-PE, HLA-DR-PE following the manufacturer&#39;s instructions21. Incubate for 15-20 min in the dark at room temperature.</li>
	<li>Centrifuge the stained cells at 300 x g for 5 min at 4 &#176;C, aspirate the supernatant with a pipette, and wash once with cell staining buffer.</li>
	<li>Repeat the centrifugation and resuspension of the cells with 300 &#181;L of the cell staining buffer.</li>
	<li>Run the cells through the flow cytometer and analyze the antibody-stained cells according to the manufacturer&#39;s instructions.</li>
</ol>
5. Determination of cell growth curve by cell counting method
<ol>
	<li>Prepare a single-cell suspension of P5 cells using two methods by diluting them in a human MSC culture medium.</li>
	<li>Seed 8,000 cells per well in a 24-well plate, with a total of 21 wells of each method being seeded. 
	NOTE: Mix the cells gently by blowing and suctioning with pipettes before seeding.</li>
	<li>Harvest cells from three wells after 24 h of seeding. Perform cell counting using a hemocytometer to calculate the mean value. 
	NOTE: Shake the plate before the cell counting. The duration of shaking can be extended to prevent the formation of cell aggregates.</li>
	<li>Repeat the cell counting every 24 h for a total duration of 7 days. Plot a growth curve using time (d) on the X-axis and cell count (x104/mL) on the Y-axis.</li>
</ol>

Dissection and Isolation of WJ-MSCs from the Human Umbilical Cord

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The authors report no conflicts of interest.

MSCs represent a dynamic area of research with profound implications for regenerative medicine22. Their unique properties make them a focal point for scientific inquiry and hold the potential to revolutionize the treatment of a wide range of diseases and injuries7. WJ-MSCs are a distinct subset of MSCs, which can be obtained from the gelatinous connective tissue within the UC situated between the intervascular and amniotic epithelium23. The clinical ...

Since the first isolation of Mesenchymal Stem Cells (MSCs) from Wharton&#39;s jelly (WJ) in 1991, these multipotent stem cells have gained significant attention from researchers due to their regenerative properties and multilineage differentiation capacity1. MSCs can be isolated from various sources, including bone marrow, peripheral blood, dental pulp, adipose tissue, fetal (human abortion), and birth-related tissues2. The umbilical cord (UC) has emerged as a promising reservoir due to its non-invasive nature, abundant cell yield and differentiation capacity, exhibiting a high rate of proliferation, differentiation potential, and immune modulation properties3. Fetal MSCs exhibit strong stemness and immune properties, making them the primary focus of clinical trials and basic research conducted over the past two decades2,4,5. UC-derived MSCs have superior therapeutic potential compared to other sources of MSC, such as bone marrow or adipose tissue6,7.
The UC is composed of amniotic epithelium, three vessels (two arteries and one vein), and the gelatinous substance known as WJ3. Intriguingly, the UC constitutes a simple vasculature, consisting only of the endothelium and mesothelium, but not the tunica adventitia; the WJ does not contain lymph or nerves8. The UC presents a unique structure ideal for segmental separation. UC-MSCs are primarily located in the WJ. MSCs could be isolated from different compartments of the WJ, including amnion, subamnion (the amnion and subamnion also designated as cord lining region), and the perivascular area of the WJ8. Each region of the WJ has its own structure, immunohistochemical characteristics, and function3,6.
MSCs isolated from the WJ of the UC are widely regarded as having superior clinical utility compared to those from other regions3. WJ-MSCs have been extensively studied in preclinical and clinical settings for the treatment of various diseases due to their multi-line differentiation potential, immunomodulatory properties, paracrine effects, anti-inflammatory effects, and immune-privileged properties2,3. WJ-MSCs have been proven to hold promise in treating a range of diseases, including graft-versus-host disease (GvHD), graft rejection, Crohn&#39;s disease, autoimmune diseases, and cardiovascular diseases9,10,11,12,13,14. As clinical demand for WJ-MSCs continues to increase, the shortage supply of umbilical cords is currently an impediment to their widespread applications.
The yield of WJ-MSCs is dependent on the method used for cell extraction15. While WJ-MSCs can be isolated through explant culture or enzyme digestion, the latter method has a longer propagation time that may increase the risk of cell damage and decrease cell viability16. However, numerous studies have shown that the explant culture method increases cell yields and viability, and that paracrine factors released from explant tissues also help promote cell proliferation17,18.
This study applied a unique dissection approach to obtain whole WJ, yielding MSCs with enhanced proliferative capacity, viability, and quantity, while minimizing damage to the WJ. This innovative method offers a streamlined strategy for isolating WJ-MSCs, addressing critical needs in MSC applications.

<table><tbody><tr><td>APC anti-human CD44 Antibody&amp;nbsp;</td><td>Biolegend&amp;nbsp;</td><td>338806</td><td></td></tr><tr><td>24-well cell culture plates</td><td>Thermo Scientific</td><td>142475</td><td></td></tr><tr><td>APC anti-human CD73 (Ecto-5&#039;-nucleotidase) Antibody&amp;nbsp;</td><td>Biolegend&amp;nbsp;</td><td>344006</td><td></td></tr><tr><td>APC Mouse IgG1, &amp;kappa; Isotype Ctrl (FC) Antibody</td><td>Biolegend&amp;nbsp;</td><td>400122</td><td></td></tr><tr><td>Autoclave</td><td>HIRAYAMA</td><td>HVE-50</td><td></td></tr><tr><td>Automatic Cell Counter</td><td>Countstar</td><td>FL-CD</td><td></td></tr><tr><td>BAMBANKER Cryopreservation Solution</td><td>Wako</td><td>302-14681</td><td></td></tr><tr><td>Cell Staining Buffer</td><td>Biolegend&amp;nbsp;</td><td>420201</td><td></td></tr><tr><td>Centrifugal Machine</td><td>Eppendorf</td><td>5424R</td><td></td></tr><tr><td>Clean Bench</td><td>Shanghai ZhiCheng</td><td>C1112B</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Thermo Scientific</td><td>HERAcell 150i</td><td></td></tr><tr><td>D-PBS</td><td>Solarbio</td><td>D1040</td><td></td></tr><tr><td>Electro- thermostatic Blast Oven</td><td>Shanghai JingHong</td><td>DHG-9423A</td><td></td></tr><tr><td>FITC anti-human CD105 Antibody&amp;nbsp;</td><td>Biolegend&amp;nbsp;</td><td>323204</td><td></td></tr><tr><td>FITC anti-human CD90 (Thy1) Antibody&amp;nbsp;</td><td>Biolegend&amp;nbsp;</td><td>328108</td><td></td></tr><tr><td>FITC Mouse IgG1, &amp;kappa; Isotype Ctrl (FC) Antibody</td><td>Biolegend&amp;nbsp;</td><td>400110</td><td></td></tr><tr><td>Flow Cytometry</td><td>Beckman</td><td>CytoFLEX</td><td></td></tr><tr><td>hemocytometer</td><td>Superior Marienfeld</td><td>640410</td><td></td></tr><tr><td>Intracellular Staining Permeabilization Wash Buffer (10&amp;times;)&amp;nbsp;</td><td>Biolegend&amp;nbsp;</td><td>421002</td><td></td></tr><tr><td>Inverted Biological Microscope</td><td>ZEISS</td><td>Axio Vert. A1</td><td></td></tr><tr><td>Liquid Nitrogen Storage Tank</td><td>Thermo Scientific</td><td>CY50935-70</td><td></td></tr><tr><td>Normal saline (NS)</td><td>Meilunbio</td><td>MA0083</td><td></td></tr><tr><td>PBS</td><td>Solarbio</td><td>P1032</td><td></td></tr><tr><td>PE anti-human CD11b Antibody</td><td>Biolegend&amp;nbsp;</td><td>393112</td><td></td></tr><tr><td>PE anti-human CD19 Antibody&amp;nbsp;</td><td>Biolegend&amp;nbsp;</td><td>392506</td><td></td></tr><tr><td>PE anti-human CD34 Antibody</td><td>Biolegend&amp;nbsp;</td><td>343606</td><td></td></tr><tr><td>PE anti-human CD45 Antibody</td><td>Biolegend&amp;nbsp;</td><td>368510</td><td></td></tr><tr><td>PE anti-human HLA-DR Antibody&amp;nbsp;</td><td>Biolegend&amp;nbsp;</td><td>307606</td><td></td></tr><tr><td>PE Mouse IgG1, &amp;kappa; Isotype Ctrl (FC) Antibody</td><td>Biolegend&amp;nbsp;</td><td>400114</td><td></td></tr><tr><td>PE Mouse IgG2a, &amp;kappa; Isotype Ctrl (FC) Antibody</td><td>Biolegend&amp;nbsp;</td><td>400214</td><td></td></tr><tr><td>Precision Electronic Balance</td><td>Satorius</td><td>PRACTUM313-1CN</td><td></td></tr><tr><td>Snowflake Ice Machine</td><td>ZIEGRA</td><td>ZBE 30-10</td><td></td></tr><tr><td>steriled 50 mL plastic tube</td><td>Greniner</td><td>227270</td><td></td></tr><tr><td>Thermostatic Water Bath</td><td>Shanghai YiHeng</td><td>HWS12</td><td></td></tr><tr><td>Trypsin 1:250</td><td>Solarbio</td><td>T8150</td><td></td></tr><tr><td>UltraGRO-Advanced</td><td>Helios&amp;nbsp;</td><td>HPCFDCGL50</td><td></td></tr><tr><td>Ultrapure and Pure Water Purification System</td><td>Milli-Q</td><td>Milli-Q Reference</td><td></td></tr><tr><td>Xeno-Free Human MSC Culture Medium</td><td>FUKOKU&amp;nbsp;</td><td>T2011301</td><td></td></tr></tbody></table>

isolation of umbilical cord-derived mesenchymal stem cells with high yields and low damage

Mesenchymal stem cells (MSCs) are a population of multipotent cells with remarkable regenerative and immunomodulatory properties. Wharton's jelly (WJ) from the umbilical cord (UC) has gained increasing interest in the biomedical field as an outstanding source of MSCs. However, challenges such as limited supply and lack of standardization in existing methods have arisen. This article presents a novel method for enhancing MSC yield by dissecting intact WJ from the umbilical cord. The method employs blunt dissection to remove the epithelial layer, maintaining the integrity of the entire WJ and resulting in an increased quantity and viability of harvested MSCs. This approach significantly reduces WJ waste compared to conventional sharp dissection methods. To ensure the purity of WJ-MSCs and minimize external cellular influence, a procedure utilizing internal tension to peel off the endothelium after flipping the UC was conducted. Additionally, the Petri dish was inverted for a short time during explant culture to improve attachment and cell outgrowth. Comparative analysis demonstrated the superiority of the proposed method, showing a higher yield of WJ and WJ-MSCs with better viability than traditional methods. The similar morphology and expression pattern of cell surface markers in both methods confirm their characterization and purity for various applications. This method provides a high-yield and high-viability approach for WJ-MSC isolation, demonstrating great potential for the clinical application of MSCs.

The procedures for collecting and culturing UC-MSCs, as well as their subsequent analysis, are summarized in Figure 1. The UC was neatly dissected into several sections using the unique method; the specific operation diagram of the main procedures is illustrated in Figure 2. The outgrowth of cells from explant cultures was routinely monitored and recorded. Adherent spindle-shaped cells were observed approximately 4 days after culturing the explants and increasin...

Author Spotlight: Advancements in Stem Cell-Derived Vascularized Organoids and Optimized Isolation of Wharton's Jelly for Enhanced MSC Production

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

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

Mesenchymal stem cells (MSCs) are a population of multipotent cells with remarkable regenerative and immunomodulatory properties. Wharton's jelly (WJ) ...

isolation-umbilical-cord-derived-mesenchymal-stem-cells-with-high

Research

JoVE Journal

Medicine

1.7K Views.  Beijing University of Chinese Medicine. This study presents a unique blunt dissection procedure to preserve the integrity of Wharton's jelly (WJ), resulting in less damaged WJ and a greater quantity and viability of the harvested mesenchymal stem cells (MSCs). The method demonstrates superior yield and proliferative ability compared to conventional sharp dissection methods.

Video: Author Spotlight: Advancements in Stem Cell-Derived Vascularized Organoids and Optimized Isolation of Wharton's Jelly for Enhanced MSC Production

Cholesterol Efflux Assay

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of cellular membranes, apoptosis and necrosis 1. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare 2. Excessive cholesterol is more common problem 3. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I. 
The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required.
The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.

The cholesterol assay is designed to quantitate the rate of cholesterol efflux from cultured cells and the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of labelling cells with cholesterol, equilibration of cholesterol among intracellular pools and release of cholesterol to an extracellular acceptor.

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of ...

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 ...

Developmental Biology

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

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

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

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Biology

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 ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

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

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

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).

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 ...

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 ...

Functional Analysis of BAMBIhighMFGE8high UC-MSC Subpopulations

Isolation of Human BAMBIhighMFGE8high Umbilical Cord-Derived Mesenchymal Stromal Cells

Umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) present low immunogenicity and potent immunomodulatory effects for treating various diseases. Human UC-MSCs are a heterogeneous population consisting of three main subpopulations with different cell shapes, proliferation rates, differentiation abilities, and immune regulatory functions. Previously, BAMBIhighMFGE8high UC-MSCs, the first subgroup successfully isolated from UC-MSCs were found to fail to alleviate lupus nephritis. Hence, the function and underlying mechanism of this subgroup in MSC therapy for diseases remains unknown. It is necessary to isolate and further investigate BAMBIhighMFGE8high UC-MSCs in terms of their phenotype, metabolism, and function to completely understand the nature of this MSC subgroup. In this protocol, we describe a detailed method for isolating the BAMBIhighMFGE8high subpopulation from human UC-MSCs. The subpopulation of UC-MSCs is labeled with two surface markers, BAMBI and MFGE8, by flow cytometry sorting. The isolated cells are cultured and verified by flow cytometry analysis. The specific genes expressed in the BAMBIhighMFGE8high UC-MSCs are identified by RT-qPCR. This protocol results in highly efficient and pure cell sorting and describes the marker profiles of the BAMBIhighMFGE8high UC-MSCs.

The isolation of BAMBIhighMFGE8high MSCs, one of the three main subgroups constituting heterogeneous human UC-MSCs, is helpful for fully understanding the characteristics and functions of this subtype for its future application to improve clinical efficacy in specific diseases. Here, we present a method for sorting BAMBIhighMFGE8high UC-MSCs.

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

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

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