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Chapter 17 Isolation, Propagation, and Characterization of Human Umbilical Cord Perivascular Cells (HUCPVCs) Rahul Sarugaser, Jane Ennis, William L. Stanford, and John E. Davies Abstract Current sources of mesenchymal cells, including bone marrow, fat and muscle, all require invasive procurement procedures, and provide relatively low frequencies of progenitors. Here, we describe the non-invasive isolation, and characterization, of a rich source of mesenchymal progenitor cells, which we call human umbilical cord perivascular cells (HUCPVCs). HUCPVCs show a similar immunological phenotype to bone marrow-derived mesenchymal stromal cells (BM-MSCs), since they are non-alloreactive, exhibit immunosuppression, and significantly reduce lymphocyte activation, in vitro. They present a non-hematopoietic myofibroblastic mesenchymal phenotype (CD45-, CD34-, CD105+, CD73+, CD90+, CD44+, CD106+, 3G5+, CD146+); with a 1:300 frequency at harvest, a short-doubling time, and a clonogenic frequency of >1:3 in culture. Furthermore, in addition to robust quinti-potential differentiation capacity in vitro, HUCPVCs have been shown to contribute to both musculo-skeletal and dermal wound healing in vivo. Key words: Mesenchymal progenitor cell, mesenchymal stem cell, umbilical cord.

1. Introduction Mesenchymal progenitor cells (MPCs), or mesenchymal stem/ stromal cells (MSCs) have classically been obtained from bone marrow (BM), and more recently from adipose and muscle tissue. Although these cells have been shown to differentiate in vitro into various mesenchymal lineages, a major limitation is availability. The harvest of BM is a highly invasive procedure, and the number and maximal life span of BM-MSCs declines with the increasing age of the donor (1). The use of alternative MSC sources is thus required for the large-scale expansion necessary to produce the number of cells needed for clinical applications. Here we describe a unique, easily harvested, rapidly proliferating, human umbilical Julie Audet, William L. Stanford (eds.), Stem Cells in Regenerative Medicine: Methods and Protocols, vol. 482 Ó 2009 Humana Press, a part of Springer ScienceþBusiness Media DOI 10.1007/978-1-59745-060-7_17 Springerprotocols.com

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cord perivascular cell (HUCPVC) population that provides high yields of MSCs (2). HUCPVCs are acquired through digestion of Wharton’s jelly of the human umbilical cord. The unique dissection procedure specifically targets the perivascular cells, yielding a high-colony forming unit-fibroblast (CFU-F) frequency of 1:300, significantly higher than that observed in either neonatal bone marrow (1:104) (1) or umbilical cord blood (1:5108) (3). These highly proliferative cells exhibit a doubling time of 20 hr (serum-dependent), and a >1:3 clonogenic frequency in culture. HUCPVCs present a myofibroblastic phenotype in culture, expressing high levels of -smooth muscle-actin, desmin, vimentin, and the pericyte marker 3G5 (4). HUCPVCs have been characterized as CD45-, CD34-, CD105+, CD73+, CD90+, CD44+, CD106+, CD49e+, CD73+, CD146+, Oct4-, and telomerase negative, with expression levels similar to those observed in BM-MSCs (5). Also similar to BM-MSCs (6), HUCPVCs have an immunoprivileged phenotype in vitro, in that they do not cause lymphocyte proliferation in a lymphocyte co-culture. In addition, they are also immunomodulatory, reducing lymphocyte proliferation in one- and two-way mixed lymphocyte cultures (7). Finally, HUCPVCs maintain the potential to differentiate into all five mesenchymal lineages in culture: fibroblast, bone, cartilage, fat and muscle; and, due to their >1:3 clonogenic capacity, can be induced to do so at the clonal level.

2. Materials 2.1. Umbilical Cord Dissection

1. Umbilical cord (20–40 cm) from full term birth 2. -MEM (VWR) 3. 4 oz sample container (VWR) 4. Antibiotics: Penicillin G (167 U/ml) (Sigma), Gentamicin solution (50 mg/ml) (Sigma), and Amphotericin B (0.3 mg/ ml) (Sigma) 5. HUCPVC dissecting tray with sterile surgical instruments (scissors and forceps) 6. Sterile silk sutures (VWR) 7. 50 ml polypropylene tubes (VWR) 8. Collagenase Type I (Sigma) 9. Hyaluronidase Type II (Sigma) 10. D-PBS (VWR) 11. Rotator (VWR)

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12. Ammonium chloride solution (StemCell Technologies) 13. DMSO (Sigma) 14. Fetal bovine serum (FBS) 2.2. HUCPVC Culture

1. Supplemented medium (SM): 95% -MEM, 5% FBS, and antibiotics 2. 75 cm2 tissue culture-treated flask (Falcon) 3. 0.25% trypsin solution (Gibco)

2.3. Characterization

1. D-PBS 2. FBS 3. Goat serum (Gibco) 4. Mouse–anti-human monoclonal antibodies: CD146, CD105 (SH2), CD73(SH3), CD90 (Thy1), CD44, CD117 (c-kit), CD34, CD45, STRO-1, 3G5, actin, desmin, vimentin, MHC I, MHC II, and Alexafluor 488 goat–anti-mouse secondary antibody (Molecular Probes)

2.4. Immunocharacterization

1. Supplemented medium (SM) (see Section 2.2.1) 2. 75 cm2 tissue culture-treated flask (Falcon) 3. 0.25% trypsin solution (Gibco) 4. 20 mL syringe (BD) 5. 40 mL heparinized whole blood (venipuncture) 6. Heparin 7. Ficoll-PaqueTM 8. D-PBS 9. WBC media (WM): 90% RMPI-1640, 10% FBS, and antibiotics (see Section 2.1.4) 10. 96-well tissue culture-treated plates (Falcon) 11. 24-well tissue culture-treated plates (Falcon) 12. TranswellTM insert for 24-well plate 13. 5-Bromo-2-deoxyuridine, and antibody 14. CD25 (interleukin-2 receptor) antibody

2.5. Mesenchymal Differentiation and Characterization

1. Supplemented medium (SM) (see Section 2.2.1). 2. 75 cm2 tissue culture-treated flask (Falcon). 3. 0.25% trypsin solution (Gibco). 4. Osteogenic induction medium: SM, 10 nM dexamethasone (Sigma-Aldrich), 5 mM b-glycerophosphate (Sigma-Aldrich), and 50 mg/ml l-ascorbic acid (SigmaAldrich).


5. Tetracycline stain: 9 mg/ml tetracycline (Sigma-Aldrich). 6. Alkaline phosphatase & Von Kossa staining: 10% NFB: 100 ml formalin/formaldehyde, 16 g Na2HPO4, and 4 g NaH2PO4.H2O in 1 L distilled water. 7. 2.5% silver nitrate solution: 2.5 g AgNO3 in 100 ml distilled water. 8. Naphthol AS MX-PO4 (Sigma). 9. N,N-dimethylformamide (DMF) (Fischer Scientific, D1191). 10. 0.2 M Tris–HCl (pH 8.3). 11. Red violet LB salt (Sigma F1625). 12. Sodium carbonate formaldehyde: 25 ml formalin/formaldehyde, 5 g Na2CO3in 100 ml distilled water. 13. Chondrogenic induction medium: SM, 10 ng/ml transforming growth factor-b 1 (TGF-b1) (Chemicon). 14. Collagen II staining: Mouse–anti-human collagen II antibody (Chemicon) and goat–anti-rabbit Alexa Fluor 488 secondary antibody (Molecular Probes). 15. Alcian blue solution (pH 2.5): 1 g 8GX Alcian blue, 3% acetic acid solution, and 0.1% nuclear fast red solution: (0.1 g nuclear fast red, 5 g ammonium sulfate, and 100 ml distilled water). 16. Adipogenic induction medium: 87% -MEM, 10% antibiotics, 3% FBS, 33 mM biotin (Sigma-Aldrich), 17 mM pantothenate (Sigma-Aldrich), 5 mM Rosiglitazone (Cayman Chemical), 100 nM bovine insulin (Sigma-Aldrich), 1 mM dexamethasone (Sigma-Aldrich), and 200 mM isobutyl methylxanthine (Sigma-Aldrich). 17. Oil Red O (Sigma-Aldrich). 18. Myogenic induction medium: 88% -MEM, 10% antibiotics, 2% horse serum (Gibco, Lot 480116), 1 nM dexamethasone (Sigma-Aldrich), and 2 mM hydrocortisone (Sigma-Aldrich). 19. Myogenic staining: rabbit–anti-human MyoD primary antibody (Santa Cruz biotech) and mouse–anti-human fast-skeletal-MLC primary antibody (Sigma-Aldrich), goat–antirabbit Alexa Fluor 488, and goat–anti-mouse Alexa Fluor 555 secondary antibodies (Molecular Probes). 20. 3.7% formalin. 21. RNA isolation and reverse transcription (RT): 0.5 ml Trizol reagent (Invitrogen); Primers: Runx2, Collagen IA1, Osteocalcin, Osteopontin, Sox9, Collagen II, Aggrecan, LPL, MyoD, Myf5, Desmin, Fast skeletal myosin light chain, and myosin heavy chain.


2.6. Clonal isolation of HUCPVCs

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1. Supplemented medium (SM) (see Section 2.2.1). 2. 75 cm2 tissue culture-treated flask (Falcon). 3. 96-well tissue culture-treated plates (Falcon). 4. 0.25% trypsin solution (Gibco).

3. Methods 3.1. Umbilical Cord Dissection for HUCPVC Extraction

1. A healthy umbilical cord is obtained from a full term delivery and placed in a 4 oz container containing -MEM and antibiotics. The cord is then transported and stored at room temperature until dissected. The remainder of the protocol is carried out aseptically (see Notes 1 and 2). 2. The cord is placed on the HUCPVC dissecting tray (see Fig. 17.1), and clamped down at one end. 3. A scalpel is used to make a circumferential incision in the amniotic epithelium close to the clamped end of the cord. The epithelium is then separated from the bulk of the cord using blunt dissection, and removed with forceps. The epithelium is usually removed in one tubular piece, although if breakage does occur, the process can be repeated to ensure all epithelium is removed. 4. The cord is then severed from the clamped end, and the three vessels longitudinally separated using forceps. These individual

Fig. 17.1. Human umbilical cord clamped onto custom-designed dissecting table which allows easy removal of the amniotic epithelium and access to the umbilical vessels. The latter are surrounded by the perivascular tissue from which HUCPVCs are harvested.


vessels are then tied in a loop using silk sutures, and placed in 40 ml of 100 U/mL Type I collagenase and 0.01 U/mL hyaluronidase in a 50 mL Falcon tube and left to digest in a rotator for 3–5 hr (37°C oven), depending on the amount of matrix on the vessels. 5. After the digestion is completed, the looped vessels are removed from the suspension and the tubes are then centrifuged at 285 g for 10 min. 6. The supernatant is then aspirated and the cell pellets pooled. The cells are treated with 50 ml ammonium chloride (0.8%) and allowed to incubate at room temperature for 5 min to lyse the erythrocytes. The tube is then centrifuged for 10 min at 285 g. The supernatant is discarded, the cells are washed and counted, and plated in the T-75 tissue culture flasks in SM. 3.2. HUCPVC Culture

1. The cells are cultured on tissue-culture treated plastic, usually T-75 flasks. Once the cells reach 80–90% confluence, they are ready to be sub-cultured. 2. For sub-culture, the cells are washed twice with PBS, 5 ml of 0.25% trypsin is added, and the cells are incubated for 2–5 min. Once the cells are in suspension, they can be centrifuged for 5 min at 285 g before removing the supernatant. The cells are then resuspended in fresh SM and counted. 3. For optimal proliferation, the cells are then seeded at a density of 4 103cells/cm2 in SM, with the SM being replaced every 2–3 days until the cells are ready for sub-culture and seeding as required.

3.3. Characterization of HUCPVCs

1. The cells are washed, resuspended as described above, and placed on ice. The cells are then permeabilized with 1 ml of methanol (only for analysis of intracellular markers). The cells are then blocked on ice in 10% FBS in PBS for 1 hr. 2. After blocking, 1–2 ml (or respective working dilution) of the primary mouse–anti-human antibody is added to the cells in 0.5% goat serum in PBS, and incubated on ice for 20 min to overnight (for low-expressing markers). The cells are then washed twice in 2% goat serum in PBS, and then incubated with the Alexafluor 488 goat–anti-mouse secondary antibody for 20 min, and washed again in 2% goat serum in PBS. 3. The cells are analyzed by flow cytometry or immunohistochemistry as is shown in Fig. 17.2 and Color Plate 13, for the -actin, desmin, and vimentin.


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Fig. 17.2. HUCPVCs expressing -actin (a), vimentin (b), and desmin (c). (See Color Plate 13)

3.4. Immuno characterization of HUCPVCs 3.4.1. Immunoprivilege

1. HUCPVCs are plated in 96-well plates at 104 cells/well. 2. Blood is obtained using heparinized syringes, and processed in a sterile biosafety cabinet. 3. Cells are separated using a Ficoll-PaqueTM density centrifugation (20 mL blood layered slowly over 20 mL Ficoll), and the leukocytes are removed from the buffy coat and counted. 4. The cells are plated in the 96-well plate at 105 cells/well. 5. The two cell populations are co-cultured for 6 days before the leukocytes are assessed for BrdU expression using flow cytometry.

3.4.2. Immunomo dulation

1. HUCPVCs are plated in 96-well plates at 104 cells/well. 2. Blood is obtained from two mismatched donors using heparinized syringes, and processed in a sterile biosafety cabinet. 3. Cells are separated using a Ficoll-PaqueTM density centrifugation, and the leukocytes are removed from the buffy coat and counted. 4. Both populations of leukocytes are plated in the 96-well plate at 105 cells/well. 5. The three cell populations are co-cultured for 6 days before the leukocytes are assessed for proliferation or co-staining with BrdU and CD25 (see Fig. 17.3). 6. This assay can also be performed using 24-well plates, seeding the HUCPVCs on a TranswellTM insert, and placing the insert in a well with the leukocytes. The same endpoints are assessed.

3.5. Mesenchymal Differentiation and Characterization

1. For CFU-F frequency, cells should be seeded into six-well plates at low dilutions ranging from less than 1 cell/well to 12 cells/well. CFU-F frequency of HUCPVCs in culture should be observed as approximately 1:3.


Fig. 17.3. HUCPVCs can differentiate into bone, cartilage, adipose, and muscle in vitro. Under induction, bone nodules are observed in culture (a). Cartilage pellet cultures of HUCPVCs express collagen II (b) and glycosaminoglycans that stained with Alcian blue (c). HUCPVC-derived adipocytes stain with Oil Red O (d), while myogenically-induced HUCPVCs expressed high levels of MyoD (e), and fast skeletal myosin light chain (FSMLC) (f) in multinucleated HUCPVC myotubes.

2. For mesenchymal differentiation analysis, the cells are subcultured and seeded into six-well plates as previously described (see Fig. 17.4 and Color Plate 14). 3. For osteogenic induction, the cells are cultured in SM until they reach 60% confluence. The medium is then replaced with

Fig. 17.4. HUCPVCs reduce lymphocyte proliferation, even if added 3 and 5 days into a 6 day culture. Addition of HUCPVCs showed a significant decrease in lymphocyte cell number compared to control (no HUCPVCs) over 6 days in a two-way MLC. There is no significant difference among HUCPVCs added on day 0, 3, or 5 (n = 6). This figure shows the average cell numbers, + standard deviations. (See Color Plate 14)


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osteogenic induction medium, which is replaced every 2–3 days for 14 days or until mineralized bone nodules can be observed. Cells are analyzed for mineralization and alkaline phosphatase expression, while RNA can be extracted for RTPCR analysis of osteogenic genes, Runx2, osteopontin, osteocalcin, and collagen I. 4. Nodules are analyzed by labeling with 9 mg/ml tetracycline overnight and examining the nodules for fluorescence. The nodules are then stained for alkaline phosphatase expression and mineralization by Von Kossa. First, the nodules are fixed in 10% cold NFB for 15 min. Fresh substrate is prepared: 0.005 g Naphthol AS MX-PO4, 200 ml DMF, 25 ml 0.2 M Tris–HCl, 25 ml distilled water, and 0.03 g red violet LB salt, and filtered with Whatman’s No. 1 filter paper immediately prior staining. Incubate for 45 min at room temperature and rinse in distilled water —three to four times. Stain with 2.5% silver nitrate for 30 min and rinse with distilled water three times. Alkaline phosphatase will appear as a red stain, while mineralized areas will appear black/grey. 5. For chondrogenic induction, the cells are resuspended as previously described, and centrifuged at 285 g for 5 min to obtain a cell pellet. The supernatant is removed and replaced with chondrogenic induction medium, which is replaced every 2–3 days for 21 days. The chondrogenic pellets are analyzed by histology for expression of collagen II and glycosaminoglycans, or by RT-PCR for expression of Sox 9, collagen II, and aggrecan mRNA. 6. Immunohistochemical analysis of collagen II expression is done as previously described. For analysis of glycosaminoglycans, alcian blue staining is used. Stain in alcian blue solution (1 g 8GX Alcian blue, 3% acetic acid adjusted to pH 2.5) for 30 min, and wash in running tap water for 2 min. Rinse in distilled water and counterstain in nuclear fast red solution for 5 min. Wash in running tap water for 1 min. Glycosaminoglycans will stain blue. 7. For adipogenic induction, the cells are cultured in SM until they reach 60% confluence. The SM is then replaced with adipogenic induction medium, which is replaced every 2–3 days for 21 days or until cells appear with large lipid droplets. Cells can be stained with 0.5% oil red O solution or analyzed by RT-PCR for expression of lipoprotein lipase (LPL). 8. For myogenic induction, the cells are cultured in SM until they reach 95% confluence. The SM is then replaced with myogenic induction medium, which is replaced every 2–3 days for 28 days or until multinucleated myotubes can be observed. Analysis of myotubes can be performed by immunohistochemical


staining for MyoD and fast-skeletal-myosin light chain, or by RT-PCR analysis of MyoD, Myf5, desmin, myosin heavy chain, and fast-skeletal-myosin light chain. 3.6. Clonal isolation of HUCPVCs

1. HUCPVCs are obtained in a single cell suspension as previously described and passed through a 70 mm filter to ensure single cell separation. 2. The cell suspension is then diluted to 1 cell/250 ml in SM. Using a multichannel micropipette, 50 ml of the suspension is deposited into individual wells of 96-well tissue culture plates, which are then incubated overnight. The following day, a further 50 ml of SM is added to the wells. The SM is then replaced every 5 days for 15 days. 3. The plates are then observed by light microscopy for the presence of cells in individual wells. Only wells with cells are continued in culture with SM replacement every 2–3 days until they reach 80–90% confluence. The cloned cells are then sub-cultured and transferred into individual wells of sixwell plates and then into T75 flasks for further expansion. The cloned cells can then be plated as required for analysis of mesenchymal differentiation potential as previously described.

4. Notes 1. All umbilical cord dissections are done aseptically in a biological safety cabinet. 2. If the umbilical cord obtained is by natural birth, it is recommended to wipe the cord with 70% alcohol three times and place in fresh MEM/antibiotic solution.

Acknowledgments The authors would like to thank the staff of the neonatal and birthing suite at Sunnybrook and Women’s College Hospital, and Mrs.Elaine Cheng for her expertise at dissecting umbilical cords.

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