Isolation and Culture of Three Kinds of Umbilical Cord Mesenchymal Stem Cells
Summary
The present protocol describes the isolation and culture of mesenchymal stem cells from the umbilical cord arteries, vein, and Wharton’s jelly.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
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 man…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| 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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