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

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

1. Place the newborn's umbilical cord into phosphate buffered saline (PBS) and transport at 4 °C.
   NOTE: Three umbilical cords from three donors were used in the present study.
2. Remove the umbilical cord blood immediately under sterile conditions, and cut along the long axis of the blood vessels in 20 cm of the umbilical cord. Separate the appropriate 20 cm of the umbilical arteries and vein, and remove the Wharton's jelly on the surface carefully.
   NOTE: Explant cultures were obtained from each region, minced into small pieces (1-2 mm³) to avoid cross contamination among the three types of stem cells.
3. Cut the umbilical arteries, vein, and Wharton's jelly to a size of 1-2 mm³ and inoculate them into 100 mm cell culture dishes. Perform the inoculation of the umbilical arteries, vein, and Wharton's jelly using tweezers following a previously published report⁵. Do not precoat the 100 mm culture plates.
   ​NOTE: The interval between the umbilical arteries, vein, or Wharton's jelly was about 1 cm. A too long collection time before separating cells may affect the cell viability and amount. It is recommended to separate the arteries and vein from the umbilical cord immediately. The collection time was up to 4 h.

2. Isolating and culturing of UCA-PSCs, UCV-PSCs, and WJ-MSCs

1. Place the cell culture dishes upside down for 3 h in a 5% CO₂ incubator at 37 °C to ensure the umbilical arteries, vein, and Wharton's jelly tissue attach to the cell culture dishes tightly before the culture medium is added.
   1. After 3 h, return the cell culture dishes to normal placement. Add 5 mL of cell culture medium (LG-DMEM, 10% FBS, 100 IU/mL of penicillin, and 100 µg/mL of streptomycin, see Table of Materials) to the cell culture dish.
2. Add 3 mL and 2 mL of the culture medium into the cell culture dish on the 3^rd and 7^th day of the first week, respectively. Be careful not to touch the tissue blocks of umbilical cord arteries, vein, and Wharton's jelly.
3. Change half of the liquid medium twice in the second week and the entire liquid medium twice in the third week.
4. Observe the cells crawling out of the tissue blocks at approximately 7 days post-culture, and then discard the tissue blocks when the cells are 80% fused at ~2 weeks.
   1. When the cells are 100% fused, passage them at a ratio of 1:3 with 2 mL of 0.05% trypsin after centrifugation at 600 x g for 4 min at room temperature.
      NOTE: The primary cells isolated from an umbilical cord were about 2 x 10⁶ UCA-PSCs, 1 x 10⁶ UCV-PSCs, and 2 x 10⁶ WJ-MSCs. The 2 x 10⁶ UCA-PSCs, 1 x 10⁶ UCV-PSCs, and 2 x 10⁶ WJ-MSCs were isolated from 20 cm of umbilical cord. The cells crawled out in all three umbilical cords, which indicated results between isolations were repeatable.
   2. Seed the cells, maintaining a concentration of 5 x 10⁵/100 cm² in the culture dish. Preserve the 5 x 10⁵ cells in 1 mL of cryopreservation solution (90% FBS, 10% DMSO). Third- to fifth-passage cells were used in the present study.

3. Detection of MSC surface markers by flow cytometry

1. Digest the third-generation cells with 0.05% trypsin to obtain a single-cell suspension. Centrifuge the cells at 600 x g for 4 min at room temperature and discard the cell supernatant using a Pasteur pipette. Then resuspend 1 x 10⁵ cells in 100 µL of PBS (1% FBS). Asses the cell number by counting the cells using a hemocytometer following a previously published report⁸.
2. Incubate the 1 x 10⁵ cells with various phycoerythrin antibodies against CD13 (FITC, 0.1 mg/mL), CD34 (FITC, 0.1 mg/mL), CD45 (FITC, 0.1 mg/mL), CD73 (FITC, 0.1 mg/mL), and HLA-DR (FITC, 0.1 mg/mL) in the dark for 30 min. For controls, incubate 1 x 10⁵ cells with FITC-IgG (FITC Mouse Anti-Human IgG, 0.1 mg/mL) (see Table of Materials).
3. Centrifuge the cells at 600 x g for 4 min at room temperature, and then discard the cell supernatant using a Pasteur pipette. Resuspend 1 x 10⁵ cells in 500 µL of PBS (1% FBS), detect by flow cytometry, and analyze by FlowJo² (see Table of Materials).

4. Differentiation of UCA-PSCs, UCV-PSCs, and WJ-MSCs into adipocytes

1. Inoculate the third-generation cells into 24-well plates at a density of 6 x 10⁴ cells/cm² with 500 µL of cell medium (LG-DMEM, 10% FBS, 100 IU/mL penicillin, and 100 µg/mL streptomycin, see Table of Materials) per well in a 5% CO₂ incubator at 37 °C.
2. Change the cell medium after 24 h, and change the control group to cell medium (LG-DMEM, 10% FBS, 100 IU/mL penicillin, and 100 µg/mL streptomycin). Conduct the experiments after the cells are 80% fused in 24-well plates.
   NOTE: The cells in the groups that induced adipogenesis were changed to an adipogenic induction medium (adipogenic differentiation kit, see Table of Materials). The medium of the cells in both groups was changed every 3 days.
3. Observe lipid droplets in cells at approximately 21 days post-induction in the adipogenic induction group and then terminate the adipogenic induction.
   NOTE: The adipogenic medium was replaced by cell culture medium to terminate the adipogenic induction^2,5. In addition, the cells in the control group showed no significant change.
4. Wash the cells three times with PBS for 5 min and then fix with 4% paraformaldehyde for 15 min at room temperature. Then, wash the cells three times with PBS for 5 min each.
5. Discard the PBS and dry the cells at room temperature for approximately 20 min. Then, add 500 µL of oil red O solution (0.5%) (see Table of Materials) to the well for 10 min.
6. Remove the oil red O solution and wash the cells with PBS three times for 5 min each. Observe the cells under a microscope.

5. Differentiation of the stem cells into osteoblasts

1. Place 500 µL of 0.1% gelatin (see Table of Materials) per well in 24-well culture plates and discard it after 30 min.
2. Inoculate 6 x 10⁴/cm² of third-generation cells into 24-well culture plates, and add 500 µL cell culture medium or osteogenic induction medium (Osteogenic Differentiation Kit, see Table of Materials) to each well in a 5% CO₂ incubator at 37 °C. In the control group, change the cell culture medium every 3 days, while also replacing the osteogenic induction medium in the osteogenic differentiation group every 3 days.
3. After 3-4 weeks, check for black calcium deposition in the cells. Wash the well with PBS three times for 5 min each.
4. Fix the cells at room temperature with 4% paraformaldehyde for 15 min and wash with PBS three times for 5 min each. Add alizarin red staining solution (1%) to the well for 20 min at room temperature, and then discard it.
5. After drying for 30 min at room temperature, observe the cells under a microscope.

6. Differentiation of the stem cells into neurons

1. Inoculate the 6 x 10⁴/cm² third-generation cells into 24-well culture plates, where sterile cover slides treated with L-polylysine were preplaced. Place the cell climbing sheets (see Table of Materials) in the 24-well plates to differentiate the cells into neurons. Culture the cells on the cell climbing sheets.
2. Then, add 500 µL of cell culture medium to the well, and replace the cell culture medium with 500 µL of neurogenic induction medium⁹ (see Table of Materials) after 24 h.
3. After induction for 24 h, add 10^-7 mol/L of ATRA and 10 ng/mL of bFGF (see Table of Materials) to the solution to maintain the induced differentiation of neurons.
4. Terminate the induction when obvious cellular synapses are formed. Check for the obvious cellular synapses using microscopy technique (see Table of Materials).
   NOTE: Immunofluorescence staining was used to identify neuron-specific markers of neuron-specific enolase (NSE, see Table of Materials), which is a marker of neurons².
5. Perform confocal imaging on the cell climbing sheets.

7. Immunofluorescence staining

1. After discarding the cell culture solution, wash the cells with 500 µL of PBS three times for 5 min each.
2. Then, add 500 µL of 4% paraformaldehyde to the well, and fix the cells at room temperature for 45 min.
3. Wash the cells with PBS three times for 5 min each.
4. Permeabilize the cells with 100 µL of 0.5% Triton-X at room temperature for 5 min.
5. Wash the cells with 500 µL PBS with 1% BSA three times for 5 min each.
6. Incubate the cells with 500 µL of PBS with 3% BSA and 0.1% Triton X-100 per well at 37 °C for 45 min to block the nonspecific binding site.
7. Dilute the primary antibody against NSE (1:100, see Table of Materials) with PBS (1:100) containing 1% BSA and 0.1% Triton X-100, and incubate the cells with the primary antibody overnight at 4 °C.
8. After discarding the primary antibody, wash the cells with 500 µL of PBS containing 1% BSA three times for 5 min each.
9. Dilute the fluorescent secondary antibody (goat Anti-Mouse IgG H&L, Alexa Fluor 488, see Tabel of Materials) with PBS (1:200) containing 1% BSA and 0.1% Triton X-100 at 1:200, and incubate the cells with the secondary antibody at 37 °C in the dark for 60 min.
10. Wash the cells with 500 µL of PBST (PBS containing 0.1% Tween-20) per well three times in the dark for 5 min each.
11. Then, add 200 µL of DAPI staining (PBST diluted, 0.1 µg/mL) to the well to stain the nucleus at room temperature without light for 5 min.
12. Wash the cells with 500 µL of PBST three times in the dark for 5 min each.
13. Add the anti-fluorescence quenching agent to the slide and then gently cover the slide with a cover glass. Add nail polish drops around the cover glass to fix it.
14. Observe the glass with a laser confocal microscope. Use the following microscopy settings: green channel with 488 nm laser and DAPI channel with 405 nm.

8. Proliferation assay

1. Seed 2 x 10³ cells per well into 96-well plates. Add 100 µL of cell culture medium into each well.
2. Add 10 µL of Cell-counting kit-8 (CCK-8, see Table of Materials) into each well.
3. After 2 days of incubation, measure the OD value at 450 nm on a microplate reader on days 1-7.
4. Analyze the OD values of the different types of stem cells using statistical software (see Table of Materials) and present them as means ± SD. Perform one-way analysis of variance (ANOVA). Consider P value < 0.05 to be statistically significant.

Results

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-atta...

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...

Materials

Name	Company	Catalog Number	Comments
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