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 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.
Human umbilical cord mesenchymal stem cells (UC-MSCs) are widely used in regenerative medicine because of their noninvasive operation, low immunogenicity, and lack of ethical dispute1. In many studies, UC-MSCs isolated from Wharton’s jelly (WJ) can attach to the wall, undergo multi-differentiation, and express markers of mesenchymal stem cells (MSCs)2. However, almost all MSCs originate from the perivascular region3. Pericytes, as a subset of perivascular cells, are progenitor cells of MSCs4. Therefore, UC-MSCs can be isolated from the umbilical cord WJ, umbilical arteries (UCAs), and umbilical vein (UCV), known as UCA-PSCs, UCV-PSCs, and WJ-MSCs, respectively5. This method aimed to isolate and culture the three different types of stem cells. The isolation and culture of UCA-PSCs, UCV-PSCs, and WJ-MSCs are very important to provide more sources of MSCs.
The present study describes the isolation, culture, and future application of UCA-PSCs, UCV-PSCs, and WJ-MSCs, which have cellular adhesion, express the markers of MSCs, and have multidirectional differentiation. The isolated stem cells were observed under microscopy and subjected to cell culture, cell passage, cell cryopreservation, and cell recovery. The rationale behind the use of this technique was cells crawling out from tissue. Compared to the previous method, such as flow cytometry or immunomagnetic bead techniques, which were complex and expensive6, the US-MSCs can be massively isolated by the adherent separation and cell crawling method; these were used in the previous study5. Flow cytometry analysis was performed on the derived stem cells to detect whether these cells express MSC markers. Multidirectional differentiation of the stem cells was introduced to detect whether the three kinds of cells have the potential to differentiate into adipocytes, osteoblasts, and neuroblasts. The isolation and culture of three types of stem cells from the umbilical cord were important in clinical use and helpful for researchers for diverse future applications.
All experimental procedures were approved by the Clinical Research Ethics Committee, Third Affiliated Hospital, Soochow University. Informed written consent was obtained from the human subjects. Individuals with full-term vaginal delivery or cesarean section were included in the present study to obtain the umbilical cord. The umbilical cord comes from healthy newborns without gender bias. The neonate had an Apgar score of 8-10. The Apgar score is a quick test for newborns given soon after their birth. This test checks a baby's muscle tone, heart rate, and other signs to see if any additional medical or emergency care is needed7. On the other hand, patients with major diseases, such as heart, liver, kidney, or other infectious diseases, were excluded from the present study.
1. Collection of human umbilical cord
2. Isolating and culturing of UCA-PSCs, UCV-PSCs, and WJ-MSCs
3. Detection of MSC surface markers by flow cytometry
4. Differentiation of UCA-PSCs, UCV-PSCs, and WJ-MSCs into adipocytes
5. Differentiation of the stem cells into osteoblasts
6. Differentiation of the stem cells into neurons
7. Immunofluorescence staining
8. Proliferation assay
Isolation and culture of UCA-PSCs, UCV-PSCs, and WJ-MSCs from the umbilical cord
The umbilical arteries, umbilical vein, and Wharton's jelly were mechanically separated from the umbilical cord and cut into 2-3 cm3 pieces. The distance between arteries, vein, or Wharton's jelly tissue blocks was approximately 1 cm, arranged in a quincunx shape (Figure 1A–C). The three kinds of stem cells were isolated by a tissue-attached culture method5. After approximately 1 week of attached culture, the cells were observed to crawl out radially, exhibiting a long spindle shape and rapid proliferation (Figure 1D–F). The cells reached 70%-80% confluence at approximately 2 weeks, and the growth rate was accelerated after passage. The cells either grew in an even spindle, parallel arrangement, or vortex growth (Figure 1G–I). In addition, the different stem cells had similar proliferation tendencies as demonstrated by the CCK-8 assay (Supplementary Figure 1). However, on days 4 and 5, UCA-PSCs had a significant higher growth rate compared to UCV-PSCs (P < 0.05).
UCA-PSCs, UCV-PSCs, and WJ-MSCs expressed MSC surface markers
The surface markers of third-generation stem cells were identified by flow cytometry. As shown in Figure 2A–O, UCA-PSCs, UCV-PSCs, and WJ-MSCs all highly expressed the MSC-specific surface markers CD13 and CD73, but did not express CD34 and CD45, which are endothelial cell markers and hematopoietic stem cell markers. The cells did not express HLA-DR, a hematopoietic stem cell marker2. The characterization of Adipose-derived stem cells (ADSCs) as a control was displayed in Supplementary Figure 2A-E, which indicates that the stem cells expressed the surface marker of MSCs consistent with that of ADSCs. MSCs were incubated with FITC-IgG and were negative for the FITC signal (Supplementary Figure 3A). In addition, the results of three repeated experiments were analyzed in Supplementary Figure 4, which proved the method was reproducible.
UCA-PSCs, UCV-PSCs, and WJ-MSCs possessed adipogenic differentiation potential
After adipogenic induction, the morphology of the isolated stem cells changed from long spindle to round. After approximately 12 days of adipogenic induction, round lipid droplets were observed in the cells, which had strong refraction. Some lipid droplets fused to form large lipid droplets. On approximately the 14th day, the three kinds of cells showed red lipid droplets of different sizes by oil red O staining (Figure 3A-C). The characterization of ADSCs as a control was displayed in Supplementary Figure 2F, which indicates that the different stem cells possessed adipogenic differentiation potential consistent with that of ADSCs. For adipogenic differentiation, there were no lipid droplets in the human endometrial stromal cells (ESCs) (Supplementary Figure 3B).
UCA-PSCs, UCV-PSCs, and WJ-MSCs have osteogenic differentiation potential
After osteogenic induction, the cell morphology changed from a long spindle to a lamellar structure, and the extracellular matrix began to deposit. After osteogenic induction for 10 days, calcium nodules appeared in the three kinds of cells. At approximately 21 days after induction, the cell morphology was polygonal, and a calcium nodule-like structure was seen in the center of the cells. The cells showed calcified nodules in the middle, as shown by Alizarin red staining (Figure 4A-C). The characterization of ADSCs as a control was displayed in Supplementary Figure 2G, which indicates that stem cells possessed osteogenic differentiation potential consistent with that of ADSCs. For osteogenic differentiation potential, there were no calcified nodules in the ESCs (Supplementary Figure 3C).
UCA-PSCs, UCV-PSCs, and WJ-MSCs have neurogenic differentiation potential
The three kinds of stem cells were induced by neurogenic induction solution; the spindle-shaped fibroblast-like cells became floated ball-shaped neurosphere-like cells. The cellular synapses were formed in all the cell variants. The morphology of the cells gradually became stellate and interconnected, and had nerve-like characteristics and enhanced refraction. The results of cellular immunofluorescence showed that the stem cells expressed neuron-specific markers of NSE after induction (Figure 5A-C). The results showed that the cells had multidirectional differentiation potential after induction. The characterization of ADSCs as a control was displayed in Supplementary Figure 2H, which indicates that the stem cells possessed neurogenic differentiation potential consistent with that of ADSCs. For neurogenic differentiation potential, there was no NSE signal in the ESCs (Supplementary Figure 3D).
Figure 1: Isolation and culture of cells. (A) The umbilical arteries were placed in the cell culture dish. (B) The umbilical vein was placed in the cell culture dish. (C) Wharton's jelly was placed in the cell culture dish. (D) The stem cells crawled out of the umbilical artery tissue. Scale bar = 500 µm. (E) The stem cells crawled out of umbilical vein tissue. Scale bar = 500 µm. (F) The stem cells crawled out of the Wharton's jelly tissue. Scale bar = 500 µm. (G) Morphology of cultured UCA stem cells after passage. Scale bar = 500 µm. (H) Morphology of cultured UCV stem cells after passage. Scale bar = 500 µm. (I) Morphology of cultured WJ stem cells after passage. Scale bar = 500 µm. Please click here to view a larger version of this figure.
Figure 2: Flow cytometry analysis of UCA-PSC, UCV-PSC, and WJ-MSC surface markers. (A-E) UCA-PSCs were negative for CD34, CD45, and HLA-DR. UCA-PSCs were positive for CD13 and CD73. (F–J) UCV-PSCs were negative for CD34, CD45, and HLA-DR. UCV-PSCs were positive for CD13 and CD73. (K–O) WJ-MSCs were negative for CD34, CD45, and HLA-DR. WJ-MSCs were positive for CD13 and CD73. The x-axis is the amount of fluorescence. Please click here to view a larger version of this figure.
Figure 3: Adipogenic differentiation. For adipogenic differentiation, the lipid droplets in the (A) UCA-PSCs, (B) UCV-PSCs, and (C) WJ-MSCs cultured in an adipogenic induction medium were stained with oil red O. Scale bar, 50 µm. Please click here to view a larger version of this figure.
Figure 4: Osteogenic differentiation. Calcium deposits in (A) UCA-PSCs, (B) UCV-PSCs, and (C) WJ-MSCs cultured in an osteogenic induction medium were stained with Alizarin red for osteogenic differentiation. Purple-red clumps are calcium nodules. Scale bar = 50 µm. Please click here to view a larger version of this figure.
Figure 5: Neurogenic differentiation. NSE in (A) UCA-PSCs, (B) UCV-PSCs, and (C) WJ-MSCs cultured in a neurogenic induction medium. Green shows NSE stain, and blue shows nucleus. Scale bar = 50 µm. Please click here to view a larger version of this figure.
Supplementary Figure 1: Cell proliferation. CCK-8 assays reflected that the different stem cells had similar proliferation tendencies. On days 4 and 5, UCA-PSCs had a significant higher growth rate than UCV-PSCs. The error bars represent the means ± SE of the three independent experiments performed in triplicate. P < 0.05. Please click here to download this File.
Supplementary Figure 2: Characterization of Adipose-derived stem cells (ADSCs) as the positive control. (A–E) ADSCs were negative for CD34, CD45, and HLA-DR. ADSCs were positive for CD13 and CD73. (F) For adipogenic differentiation, the lipid droplets in ADSCs cultured in an adipogenic induction medium were stained with oil red O. Scale bar = 50 µm. (G) Calcium deposits in ADSCs cultured in an osteogenic induction medium were stained with Alizarin red for osteogenic differentiation. Scale bar = 50 µm. (H) NSE in ADSCs cultured in neurogenic induction medium. Scale bar = 50 µm. Please click here to download this File.
Supplementary Figure 3: Characterization of MSCs and human endometrial stromal cells (ESCs) as the negative control. (A) MSCs were incubated with FITC-IgG and were negative for the FITC signal. (B) For adipogenic differentiation, there were no lipid droplets in ESCs. Scale bar = 50 µm. (C) For osteogenic differentiation, there were no calcium deposits in ESCs. Scale bar = 50 µm. (D) There was no NSE signal in ESCs. Scale bar = 50 µm. Please click here to download this File.
Supplementary Figure 4: Profiles of cell surface markers in the stem cells, and IgG. UCA-PSCs, UCV-PSCs, and WJ-MSCs were negative for CD34, CD45, and HLA-DR. UCA-PSCs, UCV-PSCs, and WJ-MSCs were positive for CD13 and CD73. IgG as overlay isotype control was negative for CD34, CD45, HLA-DR, CD13, and CD73. The results of three repeated experiments were analyzed, which proved the method was reproducible. The error bars represent the means ± SE of three independent experiments performed in triplicate. Please click here to download this File.
This study isolated three different kinds of cells from the umbilical cord arteries, vein, and Wharton’s jelly. The umbilical cord was delivery waste, and its use was simple, safe, and without ethical dispute5. UC-MSCs are original and have strong differentiation ability1. Previous studies have shown that the amount of UC-MSCs isolated from umbilical cords by the collagenase, trypsin, and hyaluronidase digestion method was not abundant; the stem cells cannot be passaged many times in approximately 70% of the specimens which cannot meet clinical needs10. The other separation methods of UC-MSCs, such as flow cytometry or immunomagnetic bead techniques, were complex and expensive11. The adherent separation method6 can rapidly and massively isolate UC-MSCs. Their differentiation and proliferation ability are stronger than the cells isolated by collagenase digestion in vitro12. It can be expanded to obtain sufficient MSCs quickly to meet the needs of clinical treatment13,14.
The stem cells were isolated and cultured from the umbilical cord using the adherent method. The cells were isolated from the adherent tissue and adhered to the wall as spindle-like fibroblasts. The umbilical cord arteries, vein, and Wharton’s jelly were segmented to 1 cm and placed in a cell culture dish. The cell culture dish was inverted in a cell incubator for 3 h before the cell culture medium was added2. The critical steps in the protocol were the proper size of the umbilical cord arteries, vein, and Wharton’s jelly, and the time to invert the cell culture dish. The cell dish was inverted for 3 h to make the tissue block adhere closely. After adding the cell culture medium, the tissue block was still fixed on the cell dish, which is important for the subsequent cell crawling out5. Troubleshooting of the technique involved the failure of cells to crawl out, which was due to the umbilical cord tissue leaving the body for more than 24 h15.
The limitations of this technique consisted of the risk of cell culture contaminants and failure of cell crawling out due to a long period of time for the isolation and culture16. However, the method of the isolation and culture of the stem cells used was economical and could obtain a large number of cells, which had significance with respect to existing methods5. The detection of cell surface molecular markers showed that the cells did not express CD34, CD45, or HLA-DR, but highly expressed CD13 and CD73. MSCs can be induced to differentiate into various mesoderm cells under specific conditions17; this multidirectional differentiation ability is the basis for MSCs to exert their repair function18. In this study, UCA-PSCs, UCV-PSCs, and WJ-MSCs differentiated into osteoblasts, adipocytes, and neuron-like cells under the induction of osteogenesis, adipogenesis, and neurogenesis in vitro, which is in accordance with the definition of US-MSCs19. Studies have shown that MSCs derived from different regions of the umbilical cord possess different characterisation15,16, indicating the importance of isolating the stem cells.
In recent years, there has been a new understanding of the origin of MSCs. Almost all MSCs come from perivascular tissue19. One study found that pericytes exist around the vascular region and are mainly located in capillaries and microvessels13. Pericytes express MSC markers, have multidirectional differentiation ability, and are the progenitor cells of MSCs20. Pericytes are located on the basal side of endothelial cells21. Pericytes around the vascular endothelium form a close connection with the vascular basement membrane22. Studies have shown that pericytes are the progenitor cells of MSCs, which play an important role in maintaining vascular endothelial function and vascular integrity in injury repair23. Pericytes can secrete more angiogenic factors, such as fibroblast growth factor-2 and vascular endothelial growth factor, to promote angiogenesis24. Pericytes derived from the pancreas have osteogenic properties and significantly promote angiogenesis25, which promotes the growth, development, and repair of tissue damage26. The speed and quality of angiogenesis are key factors for damage repair27. The damaged tissue must obtain enough nutrients and oxygen from the blood supply to ensure cell migration, proliferation, and differentiation28. Three different sources of stem cells have different characteristics and play different roles in the application. The previous study showed that UCA-PSCs expressed the angiogenesis related genes, CD146 and Jagged1, and had better angiogenesis ability than UCV-PSCs and WJ-MSCs5, which indicated the future applications of the technique in therapy for ischemia29.
The authors have nothing to disclose.
The authors wish to acknowledge support from the Basic Research Project of Changzhou science and Technology Bureau under grant number CJ20200110 (to YJY), the National Nature Science Foundation of China (82001629, XQS), the Youth Program of Natural Science Foundation of Jiangsu Province (BK20200116, XQS), and Jiangsu Province Postdoctoral Research Funding (2021K277B, XQS).
Adipogenic differentiation kit | Gibco | A1007001 | Multidirectional differentiation |
Alizarin red staining solution | Sigma | A5533 | Multidirectional differentiation |
Antibody against CD13 | Thermo Fisher Scientific | MA1-12034 | flow analysis |
Antibody against CD34 | BD Biosciences | 560942 | flow analysis |
Antibody against CD45 | BD Biosciences | 561865 | flow analysis |
Antibody against CD73 | BD Biosciences | 940294 | flow analysis |
Antibody against HLA-DR | BD Biosciences | 555560 | flow analysis |
Anti-fluorescence quenching agent | Abcam | AB103748 | Immunofluorescence |
Anti-Mouse IgG H&L (Alexa Fluor 488) | abcam | ab150113 | Multidirectional differentiation |
ATRA | STEMCELL Technologies | 302-79-4 | cell culture |
bFGF | Gibco | 13256029 | Multidirectional differentiation |
BSA | Sigma | V900933 | Immunofluorescence |
Cell incubator | Thermo Fisher Scientific | HERAcell 240i | cell culture |
Cell-counting kit-8 | Dojindo | CK04 | cell proliferation |
Centrifuge | Thermo Fisher Scientific | Sorvall™ MTX-150 | cell culture |
DAPI | Sigma | 10236276001 | Immunofluorescence |
DMSO | Sigma | D1435 | cell culture |
FBS | Gibco | 10099141 | cell culture |
FITC Mouse Anti-Human IgG | BD Biosciences | 560952 | flow analysis |
Flow Cytometer | Thermo Fisher Scientific | A24864 | flow analysis |
Fluorescence microscope | Thermo Fisher Scientific | IM-5 | flow analysis |
Gelatin | Sigma | 48722 | Multidirectional differentiation |
Leica Microscope | Leica | DM500 | Multidirectional differentiation |
LG-DMEM medium | Gibco | 11-885-084 | cell culture |
Microplate reader | Thermo Fisher Scientific | A51119500C | cell proliferation |
Neurogenic induction | Gibco | A1647801 | Multidirectional differentiation |
Oil red O solution | Sigma | O1516 | Multidirectional differentiation |
Osteogenic induction | Cyagen | HUXXC-90021 | Multidirectional differentiation |
Paraformaldehyde | Sangon Biotech | 30525-89-4 | Immunofluorescence |
Pasteur pipette | Biosharp | BS-XG-03L | cell culture |
PBS (phosphate buffered saline) | Hyclone | SH30256.LS | cell culture |
Penicillin streptomycin | Hyclone | SV30010 | cell culture |
Primary antibody against NSE | Santa Cruz Biotechnology | sc-292097 | Multidirectional differentiation |
SPSS 22.0 | IBM | SPSS 22.0 | Statistical analysis |
The cell climbing sheets | CITOTEST Scientific | 80346-0910 | Multidirectional differentiation |
TritonX-100 | Sangon Biotech | 9002-93-1 | Immunofluorescence |
Trypsin | Gibco | 25300120 | cell culture |
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