Generation of Mesenchymal Stem Cells from Human Umbilical Cord Tissue and their Differentiation into the Skeletal Muscle Lineage

Summary

We describe a protocol for the isolation of mesenchymal stem cells from human umbilical cord tissue and their differentiation into the skeletal muscle lineage.

Abstract

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⁻CD34⁻, 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).

Introduction

The human umbilical cord has been credited to possess a robust reservoir of mesenchymal stem cells, which are currently being explored for regenerative therapies due to their robust proliferation and differentiation rates, immunomodulatory properties, and ability to generate cells from all the three germ layers¹. The umbilical cord tissue consists of multiple compartments such as the umbilical cord blood, the umbilical vein subendothelium, and the Wharton's jelly (WJ), which in itself encompasses three indistinct regions-the perivascular zone, the intervascular zone, and the sub-amnion or the cord lining (CL)². While uMSCs can be isolated from all these different regions and broadly express key MSC markers, there is no clarity on whether these compartments contain the same population of uMSCs or display differences in their differentiation potencies³. Hence, protocols for the isolation of uMSCs require a greater precision in their mode and region of isolation, the robust characterization of differentiation potentials, and finally, a comparative analysis from different compartments of the cord.

In this context, few studies have demonstrated differences in uMSC proliferative and differentiative potentials between different parts of the cord. Of these, comparative analyses between uMSCs isolated from the CL and WJ regions revealed a greater proliferative potential in CL-derived uMSCs^3,4. In a separate study, WJ-derived uMSCs performed better in proliferation assays compared to perivascular cells (HUCPV)⁵. In examining differences between cord blood-derived uMSCs and cord tissue-derived uMSCs devoid of vascular contamination, differential expression of key MSC markers was reported between the two compartments, as well as increased proliferation rates in cord tissue-derived uMSCs⁶.

Of the several studies examining the differentiation potentials of uMSCs primarily into tissues of the mesoderm lineage such as osteogenic, adipogenic, and chondrogenic lineages, very few have provided detailed protocols for myogenic differentiation and subsequent characterization, as well as comparative analyses between various cord compartments. In this context, we have developed a robust muscle differentiation protocol and observed that cord tissue-derived uMSCs display superior myogenic differentiation capabilities compared to cord blood⁶. Here, a stepwise protocol is detailed for the isolation of uMSCs from the whole cord tissue devoid of cells associated with the vasculature, their characterization, and their differentiation into the myogenic lineage.

Protocol

The use of umbilical cord tissue in this study was approved by the Institutional Committee for Stem Cell Research (IC-SCR), the Institutional Ethics Committee, Translational Health Science and Technology Institute (IEC-THSTI), the Institutional Ethics Committee of Civil Hospital, Gurugram, Haryana, and the Institutional Biosafety Committee, THSTI. Human cord tissue samples were harvested from term deliveries at the time of birth. Informed written consent was obtained from subjects . All methods were carried out in accordance with relevant guidelines and regulations.

1. Isolation of MSCs from cord tissue

1. At the time of delivery, cut at least 5 cm of cord, preferably closer to the placenta, and sterilize the cord by swabbing the outer surface with 70% ethanol. Transfer the piece of cord tissue sequentially from one 50 mL collection tube containing phosphate-buffered saline (PBS) to another containing the same. Transport the collection tube bearing the name of the subject on ice to the laboratory within 1 h.
   NOTE: For a larger study, it might be useful to barcode the samples to enable their tracking throughout the study. Importantly, all personnel handling human tissues should be offered the full regimen of the hepatitis B vaccine.
2. In the lab, switch on the UV for 25 min in the BSL-2 hood to sterilize autoclaved instruments and pipettes prior to use.
3. Transfer the cord tissue piece from the collection tube to a 10 cm² tissue culture-treated dish containing PBS enriched with 5 g/L glucose, 50 µg/mL gentamicin, 2.5 µg/mL amphotericin B, 100 U/mL penicillin, and 100 µg/mL streptomycin (schematic in Figure 1).
4. Using a scalpel, slice the cord tissue vertically along its longitudinal axis to obtain two half cylindrical pieces. Owing to cord torsion and the mucoid surface, pin down the tissue with a pair of forceps held in the other hand.
5. At this point, observe the umbilical arteries and vein and remove the blood vessels by using a scalpel to scrape them off in one direction from the surface. Rinse the cord tissue once again in PBS to remove all residual blood associated with the tissue. Ensure that the scraping is gentle to preserve the integrity of cells in the WJ surrounding the vessels.
6. Mince each half of the cord tissue into 0.5 cm³ sized fragments and place the fragments with the luminal surface facing down on the dish. Incubate the dish briefly for 10 min in a 37 °C humidified chamber containing 5% CO₂.
7. After incubation, flood the dish containing the cord tissue pieces with 20 mL of medium containing MEM Alpha Modification without L-glutamine, ribo- and deoxyribonucleosides, 15% fetal bovine serum (not heat-inactivated), and 50 µg/mL gentamycin. Add the growth medium gently along the sides to prevent the tissue explants from being dislodged from their orientation. Add excess medium to account for a fraction that will be soaked up by the tissue explants during incubation.
8. After 3 days of incubation, add fresh medium to the cultures. Ensure that the cultures are protected from shocks and movement of the explants while handling the dishes.
9. After 1 week, remove the tissue fragments individually using sterile forceps and discard using appropriate biohazard bags for disposal. Retain the existing medium and add 10 mL of fresh growth medium. Replace the growth medium every 4 days until individual colonies reach a confluence of 70%.
   NOTE: It is likely that the cells will not be uniformly distributed throughout the dish, as there will be individual proliferative colonies that need to be monitored for confluence with time. Generally, within a month, a 10 cm² dish generates enough cells to be split into a separate dish.
10. Harvest the adherent cells using trypsin/EDTA solution (1x 0.25% trypsin and 0.02% EDTA in Hanks Balanced Salt Solution [HBSS]). Centrifuge the cell suspension at 470 × g for 5 min at 25 °C and resuspend the cell pellet in growth medium.

2. Immunophenotyping and propagation of uMSCS

1. Proceed to immunophenotyping once the adherent cells have reached 50%-60% confluence and are well spread. Do not perform MSC marker analysis on fully confluent cultures, as this tends to cause downregulation of key MSC markers.
2. After trypsinization, distribute a cell suspension of 1 × 10⁶ cells/mL in FACS tubes (1 × 10⁵ cells/tube) and stain with appropriate fluorophore-linked antibodies (all 1:50 dilution) in combination: unstained; CD105 + CD90; CD105 + CD73; CD105; CD90; CD73; CD34 + CD45 (common fluorophore); isotype controls for each fluorophore. Record a total number of events of at least 10,000 on the flow cytometer for further analysis.
   NOTE: Since the cells are analyzed for each surface marker separately, gating on cell subsets is not required.
3. In addition to the above markers, confirm the presence of positive and negative surface markers in uMSC lines created from individual cord tissue samples (Table 1).
4. Analyze the labeled cells by flow cytometry and determine the percentage of CD105⁺CD90⁺ and CD105⁺CD73⁺ cells. Analyze CD105⁺ and CD34⁻CD45⁻ cells separately.

3. Differentiation of uMSCs into skeletal muscle

1. Coat tissue culture plates with 0.01% collagen and 20 µg/mL laminin in PBS. Coat these plates for a minimum of 4 h at room temperature.
2. Plate uMSCs at a density of 10,000 cells/cm² in growth medium.
3. When the cells are 70% confluent, aspirate the growth medium and rinse the cultures 2x with PBS. Add myogenic differentiation medium (M1) comprising of DMEM + 5% horse serum + 0.1 µM dexamethasone + 50 µM hydrocortisone to the uMSCs. For determination of the kinetics of myogenic progression, add M1 medium every other day to the cultures.
4. To determine the kinetics of myogenic progression, analyze uMSCs for Pax7, MyoD, Myogenin, and MyHC expression at 2 days, 4-5 days, 6-7 days, and 10-14 days, respectively.

Results

The success of isolation of uMSCs from cord tissue is >95%, unlike the poor rates of success from whole cord blood. Upon successful isolation of uMSCs, FACS analysis reveals that all the cells are CD34⁻CD45⁻CD105⁺CD90⁺. However, in comparative analysis, uMSCs isolated from cord blood display heterogeneous populations, wherein a proportion of cells show CD34⁺CD45⁺CD105⁺ (~15%). Additionally, double-positive CD105⁺CD90

Discussion

Critical steps
A critical step in this protocol is the collection of tissue under aseptic conditions, from the time of delivery to the maintenance of sterile cultures, for the entire duration of propagation. During cord collection, it is essential that the cord does not touch any non-sterilized surface and is externally swabbed with 70% ethanol before collection in tubes containing PBS supplemented with antibiotics. It is important to limit the time between cord collection and processing of the tis...

Materials

Name	Company	Catalog Number	Comments
4',6-diamidino-2-phenylindole (DAPI)	Thermo Fisher Scientific	D1306
Amphotericin B	Sigma Aldrich	A2411
Antibiotic solution 100x Liquid, endotoxin tested (10,000 U Penicillin and 10 mg Streptomycin/mL in 0.9% normal saline)	HiMedia	A001A-50mL
Anti-GAPDH antibody	Sigma Aldrich	G8795
Anti-MyHC antibody (My32)	Novus Biologicals	NBP2-50401AF647
Anti-MyoD antibody (5.8A)	Novus Biologicals	NB100-56511
Anti-Myogenin antibody (Clone F5D)	Novus Biologicals	NBP2-34616AF594
Anti-Pax7 antibody	DSHB	DSHB-C1-576
APC Mouse anti-human CD90 clone 5E10	BD Biosciences	559869
Collagen Type 1	Merck	C8919
D (+) Glucose	Sigma Aldrich	G7021
Dexamethasone	SIGMA	D4902
FACSCanto II or FACSAria III	BD Biosciences
Fetal Bovine Serum, qualified Brazil	GIBCO	10270106	not to be heat-inactivated
FITC Mouse anti-human CD106 clone 51-10C9	BD Biosciences	551146
FITC Mouse anti-human CD14 clone M5E2	BD Biosciences	557153
FITC Mouse anti-human CD31 clone WM59	BD Biosciences	557508
FITC Mouse anti-human CD34 clone 581	BD Biosciences	555821
FITC Mouse anti-human CD45 clone HI30	BD Biosciences	555482
FITC Mouse anti-human CD49D clone 9F10	BD Biosciences	560840
FITC Mouse anti-human CD90 clone 5E10	BD Biosciences	555595
FITC Mouse anti-human HLA-A,B,C clone G46-2.6	BD Biosciences	557348
FITC Mouse anti-human IgG clone G18-145	BD Biosciences	555786
FlowJo software	BD Biosciences
Gentamicin	Sigma Aldrich	G1264
Horse serum	HiMedia	RM1239
Hydrocortisone	Merck	H4001
Laminin	Merck	L2020
MEM Alpha Modification without L-glutamine, ribo- and deoxyribonucleosides	Hyclone	SH30568.FS	Basal medium for uMSCs
PE Mouse anti-human CD105 clone 266	BD Biosciences	560839
PE Mouse anti-human CD44 clone 515	BD Biosciences	550989
PE Mouse anti-human CD49E clone llA1	BD Biosciences	555617
PE Mouse anti-human IgG clone G18-145	BD Biosciences	555787
PE-Cy7 Mouse anti-human CD73 CLONE AD2	BD Biosciences	561258
Phosphate buffered saline (PBS), pH=7.4	HiMedia	M1866
Trypsin/EDTA solution (1x 0.25% Trypsin and 0.02% EDTA in Hanks Balanced Salt Solution (HBSS)	HiMedia	TCL049-100mL