isolation and culture of wharton&#39;s jelly-derived mesenchymal stem cells (wj-mscs) from the umbilical cord

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

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.

Author Spotlight: Advancements in Stem Cell-Derived Vascularized Organoids and Optimized Isolation of Wharton&#039;s Jelly for Enhanced MSC Production

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

Our research used human stem cells to create various organoids. They can mimic human organs. We developed unique techniques for mesoderm and endoderm co-differentiation.They enable these organoids with rich blood networks. These organoids will promote organ development, regeneration medicine, disease model, drug screening, and personalized medicine, such as organoid transplantation. Our protocol addresses a research gap in optimizing the isolation of intact Wharton's jelly from the umbilical cord, while preserving its perivascular and subamniotic components.This method aims to enhance the yield, viability, and proliferative ability of mesenchymal stem cells, offering a cost-effective approach to augment MSC production. The advantage of our protocol lies in its ability to obtain a higher Wharton's jelly's yield and harvest more MSCs with better proliferative ability compared to traditional methods.

Isolation and Culture of Wharton&#39;s Jelly-Derived Mesenchymal Stem Cells (WJ-MSCs) from the Umbilical Cord

To begin, place all the sterile surgical instruments on the working platform. In the biosafety cabinet, disinfect the surface of the tube containing the previously collected human umbilical cord with 75%ethanol. Remove the sample into a Petri dish.Immerse the sample with the knots in 75%ethanol for 30 seconds. Then, in a new Petri dish, rinse the sample multiple times with sterile PBS. Using surgical scissors and forceps, cut the knots off, then cut the cord between two knots transversely into short pieces.Perfuse the vein with PBS using a one milliliter pipette until the fluid coming out of the other end of the cord becomes completely transparent. Transfer one of the already cut pieces of umbilical cord to a new Petri dish. Add the appropriate amount of PBS to keep the cord wet throughout the operation.Grasp the cord with forceps and pull out the arteries along its major axis with the help of mosquito clamps. To fix the cord horizontally with the thicker side facing up, insert one blade of the forceps into the umbilical vein and clamp it tightly, ensuring the forceps clamp on the thicker side. Using a scalpel, make a linear incision on the amniotic epithelium layer from one end to the other, along the edge of the other blade of the forceps.From the cutting gap corner, lift the corner of the amniotic epithelium with tooth clamps, and continue to cut horizontally a little further with corneal scissors along the inner surface of the epithelium. Separate the Wharton's jelly and amniotic epithelium with clamps, and gradually expand to the whole circle of one end of the umbilical cord. Peel off the epithelial layer lengthways.Insert both blades of the forceps inside the vein. Clamp a portion of Wharton's jelly, and then flip it inside out to expose the epithelium of the umbilical vein. Peel off the epithelium of the vein easily.Place the collected Wharton's jelly in a pre-labeled 90 millimeter Petri dish and cut it into one to three millimeter pieces with scissors and forceps. Invert the dish with the cap on the bottom and place it in a 5%carbon dioxide incubator at 37 degrees Celsius for 30 minutes. Change the human mesenchymal stem cell culture medium every three days.Compared to the conventional approach, the unique blunt dissection procedure showed a significant increase in Wharton's jelly yield. Further analysis by counting the total quantity of Wharton's jelly mesenchymal stem cells by both methods showed a pronounced gap in cell quantity with increasing passage numbers. The proliferation rate of mesenchymal stem cells in the novel method was significantly higher after two days.

isolation-culture-whartons-jelly-derived-mesenchymal-stem-cells-wj

Research

JoVE Journal

Medicine

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.

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.

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