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Multipotent adult progenitor cell isolation and culture procedures

2006; Elsevier BV; Volume: 34; Issue: 11 Linguagem: Inglês

10.1016/j.exphem.2006.07.013

1873-2399

April Breyer, Nima Estharabadi, Masayuki Oki, Fernando Ulloa, Molly Nelson-Holte, Laura Lien, Yuehua Jiang,

Neonatal Respiratory Health Research

We describe methods for isolation of multipotent adult progenitor cells (MAPCs) from newborn to 6-week-old mice and rats. The maintenance of these cells, including their culture, media formulas, and quality control procedures, are also explained. Additionally, ways to identify MAPCs including their phenotype and morphology are discussed. We describe methods for isolation of multipotent adult progenitor cells (MAPCs) from newborn to 6-week-old mice and rats. The maintenance of these cells, including their culture, media formulas, and quality control procedures, are also explained. Additionally, ways to identify MAPCs including their phenotype and morphology are discussed. Multipotent adult progenitor cells (MAPCs) have been previously described as bone marrow–derived cells capable of differentiating into mesenchymal, neuroectodermal, and endodermal cells [1Jiang Y. Breyer A. Lien L. Blackstad M. Verfaille C. Culture of multipotent adult progenitor cells (MAPCs).Blood. 2004; 104: 2329Crossref Scopus (77) Google Scholar, 2Jiang Y. Jahagirdar B.N. Reinhardt R.L. et al.Pluripotency of mesenchymal stem cells derived from adult marrow.Nature. 2002; 418: 41-49Crossref PubMed Scopus (5101) Google Scholar]. They have proven to be difficult to isolate and culture, so we have set out to give a detailed protocol for their isolation and maintenance. Cells are cultured on plates coated with 100 ng/mL rat fibronectin (FN; Sigma, St. Louis, MO, USA) in phosphate-buffered saline (PBS; 1×, without calcium and magnesium, Cellgro, Herdon, VA, USA). Ten-centimeter plates (Nunc, Rochester, NY, USA) are used for general maintenance, and 6- to 96-well plates (Corning, Corning, NY, USA) are used for culture as needed. Coating needs to be done for at least 1 hour at 37°C, 2 hours at room temperature, or overnight at 4°C. We have found that rat and mouse cell cultures respond better to different media formulas. For rat cell culture, the media contains 60% low glucose Dulbecco's Modified Eagle Media (DMEM) (Gibco BRL, Carlsbad, CA, USA), 40% MCDB-201 (Sigma), 1× insulin-transferrin-selenium (ITS; Sigma), 1× linoleic acid bovine serum albumin (LA-BSA; Sigma), 10−9 M dexamethasone (Sigma), 10−4 M ascorbic acid 3-phosphate (Sigma), 100 units of penicillin, 1000 units of streptomycin (Gibco), 2% fetal bovine serum (FBS; HyClone, Logan, UT, USA), 10 ng/mL human platelet-derived growth factor (R&D Systems, Minneapolis, MN, USA), 10 ng/mL mouse epidermal growth factor (Sigma), and 1000 units/mL mouse leukemia inhibitory factor (Chemicon, Temecula, CA, USA). Mouse cells are cultured in similar media with the following modifications: 1× selenium-insulin-transferrin-ethanolamine (Sigma) is used instead of ITS, a combination of 0.2 mg/mL LA-BSA and 0.8 mg/mL powdered bovine serum albumin (BSA; Sigma) is added instead of using only LA-BSA, 1× chemically defined lipid concentrate (Gibco) is included, and dexamethasone is not included. β-mercaptoethanol (1×; Gibco) is added freshly to both types of media. Media is sterilized using a 22-μm filter (Millipore, Billerica, MA, USA), and it is kept in glass bottles at 4°C for up to 2 weeks. Both cell types are very sensitive to FBS lot differences. When choosing a serum lot, testing must be done for speed of growth, correct cell morphology, expression of Oct-4, and the ability for cells to properly differentiate into the various lineages. The doubling time needs to be approximately 12 to 30 hours to ensure proper differentiation and normal cytogenetics [1Jiang Y. Breyer A. Lien L. Blackstad M. Verfaille C. Culture of multipotent adult progenitor cells (MAPCs).Blood. 2004; 104: 2329Crossref Scopus (77) Google Scholar]. Rodent MAPCs are small cells, between 9 and 12 μm in size. They are usually spindle or triangular in shape, becoming round when they are dividing (Fig. 1). Large, spread out cells with many projections are often seen but are not wanted in the cultures as these are not MAPCs. MAPCs can be derived from 1-week-old newborn mice or rats to 6-week-old rodents. The younger the rodent, the better the probability of deriving a MAPC line. The isolation procedure varies depending on the age of the rodent. Both mice and rats are euthanized using CO2, and isolation is done under sterile conditions. When sacrificing rodents, one rat or two or three mice are typically used. We have previously isolated cells from C57BL/6 and Rosa 26 mice and Sprague Dawley and Fisher rats. When sacrificing newborn rodents, the entire hind limbs are removed, and the muscles are detached from them. The bones are minced into very small pieces and placed in a 50-mL tube (Falcon, San Jose, CA, USA) with 20 to 30 mL of 0.2% collagenase (Worthington, Lakewood, NJ, USA), dissolved in Media 199 (1×, Gibco). The tube is gently shaken at 37°C for 45 minutes to an hour on a shaker. Cells are triturated by passing the cell suspension 5 to 10 times through a 23-gauge needle attached to a 10-mL syringe, resulting in a single-cell suspension. They are then passed through a 40-μm nylon mesh cell strainer (Falcon); 10% FBS is added to inactivate the collagenase, and the cells are centrifuged at 1800 rpm for 6 minutes. Cells are washed three times, using approximately 10 to 15 mL of PBS with 0.5% BSA or MAPC media each time. For adult 4- to 6-week-old rodents, the tibia and femur bones are used. It is essential that the ends of the bones are included, as it has been shown that MAPCs are located near growth plates and the endosteum and that cells from the middle part of the bone can only be cultured short term [1Jiang Y. Breyer A. Lien L. Blackstad M. Verfaille C. Culture of multipotent adult progenitor cells (MAPCs).Blood. 2004; 104: 2329Crossref Scopus (77) Google Scholar]. Again, the muscles are removed. The bones are flushed very vigorously into a 10-cm Petrie dish until they appear transparent, using a 23-gauge needle and Media 199. Approximately 15 to 20 mL of media is used in flushing the cells. Cells are triturated, filtered through a 40-μm cell strainer, and washed with PBS or media like newborn cells. The plating procedure is used for rodents of all ages. After the final washing, cells are suspended in MAPC media and counted using a hemocytometer. They are plated at 6 million cells per well on an FN-coated 6 well plate (Corning) in 2 mL media per well. After 3 days, another 1 mL of media is added to each well. For the first few weeks of culture, media is prewarmed in the 37°C incubator for about an hour before adding, so both the temperature and pH are the same as what the cells are currently being cultured in. For the rest of the first week, 1 mL of media is added every other day. At this time, many of the cells will not be attached to the plate, so media should not be removed. The second week, most of the cells will have attached, and half of the media is changed at the same time interval. Beginning the third week, cells are replated at 80% confluence, or about 2 × 104 cells/cm2. This density is typically achieved by moving six wells of cells into nine wells. Once the cells have grown to 100% confluence, they are replated at 80% confluence. After cells have been cultured for approximately a month, column depletion is done to remove CD45+ and Terr119+ cells from the culture. Following depletion, cells are kept at a significantly lower density of 1 to 2 × 102 cells/cm2. Overall cell density and limiting cell–cell contact are both important [1Jiang Y. Breyer A. Lien L. Blackstad M. Verfaille C. Culture of multipotent adult progenitor cells (MAPCs).Blood. 2004; 104: 2329Crossref Scopus (77) Google Scholar]. After depletion, cells must never be in cell–cell contact. As soon as this contact is seen, usually after about 36 to 48 hours, cells need to be split or replated. Depletion is done approximately a month after isolation using a MACS separation CS column (Miltenyi Biotec, Auburn, CA, USA) with a minimum of 2 to 3 million cells. Before adding cells, the column is washed with 30 mL of buffer, consisting of 0.5 to 1% BSA in PBS. For this, an 18- to 20-gauge needle is attached. Cells are centrifuged and then resuspended in about 80 μL of buffer. Added to this are microbeads for mouse CD45 and mouse Terr119 (Miltenyi Biotec) at 10 μL each for 10 million or fewer cells. This solution is incubated on ice for 15 to 20 minutes. After incubation, cells are washed twice, by suspending them in 10 mL of buffer and centrifuging. Following the second wash, cells are suspended in 500 μL of buffer, added to the column, and washed through in at least 30 mL of buffer. At this time, a 23- to 25-gauge needle is used. It is essential that the solution drips very slowly, approximately one drop every 2 to 3 seconds. Cells are collected in at least three separate 10-mL fractions, centrifuged, and counted. Each fraction is plated in FN-coated 96-well plates for clones to grow. A density of 10 cells/well works best for clonal growth in each well. After about 2 weeks, clones can be observed. When a colony of 30 to 50 cells with MAPC morphology is seen in a well, it is removed and plated again in one well of a 96-well plate. Clones are removed from the 96-well plates and transferred into progressively larger plates when they begin to contact each other. When enough cells have grown, quality control experiments, such as quantitative reverse-transcription polymerase chain reaction (Q-RT-PCR) and karyotyping, are performed. Every 36 to 48 hours, either the cells need to be split or the media needs to be changed. When cells are split, the media is removed and saved for trypsin deactivation. Cells are washed once with PBS. One milliliter of 0.05% trypsin (Cellgro) is added to each plate, and then the plate is tapped to detach the cells. MAPCs are small and lightly attached, so they should easily be removed from the plate quickly. Larger cells that are more strongly attached are likely to be differentiating, so short-term trypsinization gives the desired population. After tapping, cells are looked at under the microscope to see that they have detached, and then the trypsin is deactivated with the saved media. Cells are centrifuged at 1800 rpm for 6 minutes. When removing the supernatant, it is important that the pellet is not disturbed, as MAPCs are located at the top due to their small size. Cells are resuspended in 6 mL of media per 10-cm plate. Media pH is important in cell culture, as an alkaline pH will cause MAPCs to die. Therefore, it is important that plates are not removed from the incubator for extended periods of time. When media becomes pink, it is an indicator that the pH level has changed. Keeping the incubators at proper CO2 and O2 levels maintains the correct culture pH of 7.2. The CO2 level should be at 5.5 to 6%, and the O2 level is kept low, at 5%. These levels also reduce cytogenetic abnormalities [1Jiang Y. Breyer A. Lien L. Blackstad M. Verfaille C. Culture of multipotent adult progenitor cells (MAPCs).Blood. 2004; 104: 2329Crossref Scopus (77) Google Scholar]. Cells are grown at 37°C. Freezing should be done frequently, whenever there are extra cells, especially in early passages after depletion and following normal karyotype results. Cells (100,000–500,000) are frozen in each cryogenic vial (Nunc). Two medias are used for our two-step freezing process. The first consists of 80% MAPC media and 20% FBS, and the second contains 60% MAPC media, 20% FBS, and 20% dimethyl sulphoxide (Sigma). Freezing is done as quickly as possible for the highest viability. Vials are kept on ice during the freezing process. Cells are suspended in 500 μL of the first freezing media for each vial. The second step is to gently add 500 μL of the second freezing media per vial. This media is added drop by drop, and the tube is tapped or lightly shaken between drops. The solution should not be mixed by pipetting it repeatedly. Cell solution (1 mL) is added to each vial, and vials are immediately put into the −80°C freezer in a cryo 1°C freezing container (Nalgene). The vials are moved to the nitrogen tank after a minimum of 6 hours, or they can be left at −80°C overnight. The thawing procedure needs to be done quickly and gently, as cells are fragile after being frozen. Cells removed from the nitrogen tank are put on dry ice, and they are then thawed in a 37°C water bath. When the cells are partially thawed, with a small frozen crystal remaining, cells are put in 10 mL of fresh media. The suspended cells are centrifuged at 1000 rpm for 8 minutes. Cells can all be plated in one plate, or can be counted using trypan blue (Gibco) and plated at around 50,000 cells per plate. Cells grow slowly after being thawed, and therefore they are plated at a higher density than usual until the first time they are split. After 2 days, the cells are returned to the normal density of 1 to 2 × 102 cells/cm2. Levels of Oct-4 and Rex-1 are tested using Q-RT-PCR. Oct-4 levels can be low (Fig. 2c) or high (Fig. 2b) depending on culture conditions. When isolated and maintained in low-O2, approximately one-third of the clones express Oct-4 levels at 10 to 60% of ES cell levels, and Rex-1 at 5 to 30% of ES cells [3Plamqvist L. Glover C.H. Hsu L. Correlation of murine embryonic stem cell gene expression profiles with functional measures of pluripotency.Stem Cells. 2005; 23: 663-680Crossref PubMed Scopus (128) Google Scholar, 4Okamoto K. Okazawa H. Okuda A. Sakai M. Muramatsu M. Hamada H. A novel octamer binding transcription factor is differentially expressed in mouse embryonic cells.Cell. 1990; 60: 461-472Abstract Full Text PDF PubMed Scopus (607) Google Scholar, 5Scholer H.R. Ruppert S. Suzuki N. Chowdhury K. Grass P. New type of POU domain in germ line-specific protein Oct-4.Nature. 1990; 344: 435-439Crossref PubMed Scopus (591) Google Scholar, 6Rosner M.H. Vigano M.A. Ozato K. et al.A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo.Nature. 1990; 345: 686-692Crossref PubMed Scopus (754) Google Scholar]. However, MAPCs express negligible amounts of Nanog, which is expressed in ES cells [3Plamqvist L. Glover C.H. Hsu L. Correlation of murine embryonic stem cell gene expression profiles with functional measures of pluripotency.Stem Cells. 2005; 23: 663-680Crossref PubMed Scopus (128) Google Scholar, 7Mitsui K. Tokuzawa Y. Itoh H. et al.The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells.Cell. 2003; 113: 631-642Abstract Full Text Full Text PDF PubMed Scopus (2522) Google Scholar, 8Chambers I. Colby D. Robertson M. et al.Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells.Cell. 2003; 113: 643-655Abstract Full Text Full Text PDF PubMed Scopus (2595) Google Scholar]. The MAPC phenotype is evaluated using flow cytometry. High Oct-4 mouse MAPCs are negative for CD3, CD19, CD34, CD45, Gr-1, Mac-1, Sca-1, Thy-1, and major histocompatibility complex (MHC) class I and II. MAPCs have negative to very low positive levels of CD44, and they have very low positive levels of SSEA-1 and Flk-1. They are positive for c-Kit. High Oct-4 rat MAPCs are CD90 and MHC class II negative, have very low levels of MHC class I and CD44, and are CD31 positive. See Figure 2, Figure 3 for Oct-4 staining data and Figure 4, Figure 5 for fluorescein-activated cell sorting (FACS) data on high Oct-4 cells. Data for low Oct-4 cells have been previously reported [2Jiang Y. Jahagirdar B.N. Reinhardt R.L. et al.Pluripotency of mesenchymal stem cells derived from adult marrow.Nature. 2002; 418: 41-49Crossref PubMed Scopus (5101) Google Scholar].Figure 4FACS phenotype of high Oct-4 mouse MAPCs derived from green fluorescent protein transgenic mice. Red shows isotype control staining, and blue shows samples stained with antibodies. (a): Sca-1 phycoerythrin (PE) and c-kit allophycocyanin. (b): Thy1.1 PE and CD44 APC. (c): MHC-II PE and CD9 biotinylated antibody APC. (d): CD73 PE and CD45.2 biotinylated antibody APC. (e): Isotype PE and Lineage (CD3e, B220, Mac1, Gr-1, Ter-119) biotinylated antibody APC. (f): CD31 PE and Mac-1 APC. (g): GFP and CD105-purified antibody APC. (h): GFP and MHC-II PE. (i): GFP and E-cadherin-purified antibody, clone ECCD-2 (Takara Bio Inc.) APC. (j): GFP and EpCAM-purified antibody APC. APC-conjugated goat anti-rat IgG, F(ab′)2 antibody (Jackson ImmunoResearch Laboratories Inc., San Diego, CA, USA) is used as a secondary antibody (g–j). All biotinylated antibodies are followed by streptavidin (St) staining. All antibodies are from BD Pharmigen unless otherwise noted.View Large Image Figure ViewerDownload (PPT)Figure 5FACS phenotype of high Oct-4 rat cells. Red shows isotype control staining, and blue shows samples stained with antibodies. (a): CD44 fluorescein isothiocyanate (FITC) and isotype APC. (b): Isotype FITC and CD90 PerCP. (c): CD31 biotinylated antibody PE and isotype APC. (d): MHC-I biotinylated antibody PE and isotype APC. (e): MHC-II biotinylated antibody PE and isotype APC. (f): CD11b/c FITC and isotype APC. All biotinylated antibodies are followed by St staining. All antibodies are from BD Pharmigen.View Large Image Figure ViewerDownload (PPT) It is crucial that karyotyping of MAPCs is done on a regular basis to ensure that cells are cytogenetically normal. Cells are tested after depletion, once clones have been established. As cells have grown for longer amounts of time, the presence of cytogenetic abnormalities becomes more common. Therefore, they should be tested even more frequently. We suggest testing cells at least once a month. Differentiation into the various cell lineages, mesoderm, neuroectoderm, and endoderm is done after cells have grown for approximately 30 population doublings after depletion [9Reyes M. Dudke A. Jahagirdar B. Koodie L. Marker P.H. Verfaille C.M. Origin of endothelial progenitors in human postnatal bone marrow.J Clin Invest. 2002; 109: 337-346Crossref PubMed Scopus (1110) Google Scholar, 10Schwartz R.E. Reyes M. Koodie L. et al.Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells.J Clin Invest. 2002; 109: 1291-1302Crossref PubMed Scopus (1099) Google Scholar, 11Jiang Y. Henderson D. Blackstad M. Chen A. Miller R.F. Verfaille C.M. Neuroectodermal differentiation from mouse multipotent adult progenitor cells.PNAS. 2003; 100: 11854-11860Crossref PubMed Scopus (313) Google Scholar].