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The use of transgenic markers in pluripotent stem cells allows the facile isolation of transient cell populations that appear at certain phases of embryonic development. Here we describe a procedure for deriving cardiac progenitors from mouse pluripotent stem cells carrying a GFP reporter under the control of an Nkx2.5 enhancer sequence. The cells are propagated under standard conditions and differentiated using the hanging-droplet method with medium optimized for commitment to the cardiac lineage. Cardiac progenitors are isolated from the differentiation culture using fluorescence-activated cell sorting (FACS) and can be cultured further for functional characterization and experimentation. The protocols described here can be applied to both embryonic and induced pluripotent stem cells and can easily be adapted to transgenic lines carrying other cardiac cell lineage reporters.
In recent years, there has been growing interest among basic and clinical scientists in harnessing the tremendous developmental potential of pluripotent stem cells for understanding fundamental biological processes and disease pathogenesis. These cells, derived either from pre-implantation stage embryos of human or animal origin (embryonic stem (ES) cells), from neonatal or adult testis (mature adult germline stem cells (maGSC)), or by molecular reprogramming of differentiated somatic cells (induced pluripotent stem cell (iPS) cells), are believed to share similar transcriptional profiles and molecular phenotypes in culture. The ability of these cells to differentiate in vitro into all lineages of the three germ layers has allowed the isolation and phenotypic characterization of various populations of progenitor and transient-amplifying cells prior to their terminal differentiation. It is believed that by identifying and isolating lineage-specific stem/progenitor cells from pluripotent stem cells, one may then be able to translate the developmental potential of these cells into therapeutic applications for a wide range of neurological, cardiovascular, and hematological diseases. The objective of this unit is to describe, in sufficient detail, our methods for identifying and isolating cardiac progenitor cells from in vitro differentiated mouse ES and iPS cells. It is anticipated that all readers with standard training in cell and molecular biology will be able to accomplish this by following the steps described here.
Before we describe our basic protocol for isolating cardiac progenitor cells from pluripotent stem cells, it is worth mentioning the rationale and advantages for using in vitro differentiated ES or iPS cells rather than isolating them directly from developing embryos, First, working with early stage embryos can be technically difficult and costly, and the number of cells obtained may be insufficient for further experimentation. Pluripotent stem cells, on the other hand, can be expanded in large numbers to satisfy the cell number required for most studies. Second, the rapid doubling time of pluripotent stem cells allows for a short turnaround time between experiments. Third, pluripotent stem cells can be genetically modified to carry lineage specific reporters and/or mutations, which would allow for the in vitro isolation of specific cell-types of interest that recapitulate disease phenotypes without incurring the time and cost associated with generating genetically-modified animals. These advantages, coupled with the potential uses of pluripotent stem cells in cell-based therapies, account for the growing popularity of these cells in regenerative biology and medicine.
In this unit we describe the isolation and characterization of mouse ES and iPS cell-derived Nkx2.5+ cardiac progenitor cells. While a number of cardiac stem/progenitor cell populations from mouse and human ES cells have been described (Moretti et al., 2006, Kattman et al., 2006, Yang et al., 2008, Bu et al., 2009, Domian et al., 2009), the biological relationships between these cell populations remain to be clarified. Moreover, the protocols used by each laboratory to purify cardiac progenitor cells vary significantly. For example, the use of Flk1 to isolate multipotent cardiovascular progenitor cells relies on the addition of a cocktail of growth factors during differentiation (Kattman et al., 2006). When mouse ES cells are differentiated in the presence of serum and the absence of growth factor cocktail, very few cardiomyogenic Flk1+ cells can be isolated (Wu, S.M. unpublished data). Our approach here takes advantage of the availability of a committed cardiac progenitor cell marker, Nkx2.5, which has been well described to mark the first identifiable heart-forming cells in the developing embryo (Wu et al., 2006). Furthermore, we have shown that mouse ES cell-derived Nkx2.5+ cells can give rise specifically to cardiomyocytes and smooth muscle cells both in vivo and in vitro (Wu et al., 2006). Although the protocol described here is based on studies using a transgenic Nkx2.5-eGFP fluorescence reporter to identify cardiac progenitor cells, the methods for culturing, differentiating, and progenitor cell isolation can be applied to other ES and iPS cells carrying appropriate lineage or surface markers.
The first protocol describes the general culturing and in vitro differentiation of mouse ES cells by the hanging droplet method. Subsequent protocols describe the isolation, continued culture, and characterization of the FACS-purified eGFP+ cardiac progenitor cells from Nkx2.5-eGFP ES cells.
NOTE: This unit assumes that the user has some experience with the growth and maintenance of undifferentiated mouse embryonic stem (ES) cells; for example protocol, see Tremml et al., 2008.
NOTE: The following procedures are performed in a Class II biological hazard flow hood, using sterile equipment and solutions and proper aseptic technique.
NOTE: All incubations are performed in a humidified 37°C, 5% CO2 incubator unless otherwise noted.
NOTE: All centrifugations are done in an Eppendorf Model 5810R benchtop centrifuge.
This protocol is adapted from the well-described hanging-droplet method of ES differentiation (Samuelson et al., 2006), with slight modifications designed to enhance cardiac lineage differentiation (Wobus et al., 1991, Takahashi et al., 2003). It can be adapted to generate large quantities of cardiac progenitor cells by scaling up accordingly. For high-throughput screening assays, a monolayer differentiation protocol using lineage-labeled ES or IPS cells in a 96-well plate format can be used (see Alternative Protocol 1). Additionally, a hybrid protocol that incorporates both methods may be used for other applications. In such a protocol, cells differentiated in hanging-droplet cultures are dissociated at the desired time point and re-plated into 6-, 12-, 24-, or 96-well plates for further treatments and characterization (see Basic Protocol 2).
This protocol describes the dissociation of EBs into a single cell suspension to be used for flow cytometry analysis or FACS. Dissociation is also recommended for isolating particular EBs or areas in the differentiation culture for long-term culture (see Basic Protocol 4).
The Nkx2.5-eGFP marker allows for the quantization and purification of cardiac progenitor cells that develop during EB differentiation. Assessment of the efficiency of cardiac differentiation can be performed using flow cytometry to determine the percentage of cells that have committed to the cardiac lineage (based on the expression of eGFP). After cells have been prepared according to Basic Protocol 2, standard flow cytometry procedures are used to quantitate the percentage of eGFP+ cells with each batch of differentiation. A typical series of gates used for determining the percentage of eGFP+ cells present in differentiated EBs is shown in Fig. 3. We routinely observe 1–5% of eGFP+ cells present in the total PI-negative live-cell population. Fluorescence-activated cell sorting (FACS) can be used to recover the eGFP+ cells for further culture and/or experimentation (see Basic Protocol 4) if desired.
This protocol describes the continued culturing and characterization of the Nkx2.5-eGFP+ cardiac progenitor cells isolated by FACS from EB differentiation. There are a wide variety of functional assays that may be used to characterize these progenitor cells and their progeny. We will restrict our description to ones that can be performed using conventional molecular biology tools instead of more specialized techniques such as electrophysiological assessment or contractility assays of cardiomyocytes.
For experiments involving chemical or siRNA-based high-throughput screening of compounds or genes that regulate cardiac differentiation of ES or IPS cells, a monolayer differentiation assay in 96-well plate can be employed (Takahashi et al., 2003). If a fluorescence reporter is used to quantitate cardiac differentiation, a number of fluorescence-based high-throughput detection systems such as automated flow-cytometry, automated fluorescent microscopy, or automated fluorescence plate-reader, can used.
Unless otherwise noted, all solutions and media are 0.2µm-filter sterilized.
Dissolve 0.5 g of gelatin (from porcine skin) in 500ml distilled/deionized water and autoclave. Store at room temperature indefinitely.
Dispense 0.1% gelatin solution into tissue culture plates and incubate at room temperature for 15 minutes. At the end of incubation, aspirate excess gelatin solution and let the tissue culture plate surface dry for 5 minutes. Plates may be stored for at least 1–2 months at 4°C until use.
|DMEM-High Glucose GIBCO 11965-0692||500 mL|
|Non-Essential Amino Acids (10mM) GIBCO 11140-050||6.25 mL|
|L-Glutamine0^ (200mM) GIBCO 25030-081||6.25 mL|
|Penicillin/Streptomycin (10000U/ml, 10000µg/ml) GIBCO 15070-063||12.5 mL|
|Fetal Bovine Serum* GEMINI 100–106||94 mL|
|β-mercaptoethanol SIGMA M6250||4.4 µL|
|Leukemia Inhibitory Factor (107 units/mL) CHEMICON ESG1107||62.5 µL|
|L-Glutamine (200mM)||6.25 mL|
|Penicillin/Streptomycin (10000U/ml, 10000µg/ml)||12.5 mL|
|Fetal Bovine Serum* GIBCO 10437-028||94 mL|
|Ascorbic Acid (5mg/mL aqueous solution) SIGMA A4544||6.25 mL|
^L-Glutamine is very sensitive to oxidation and must be replenished every 7 days.
*FBS should be lot-tested for minimal induction of differentiation (ES media) or optimal cardiac differentiation results (differentiation media).
+Media are stable for up to 2 weeks at 4°C.
|1 M Hepes GIBCO 15630-080||2.5 mL|
|5 M NaCl||2.68 mL|
|1 M KCl||0.5 mL|
|1 M Na2HPO4||70.0 µl|
Adjust pH to 7.1 as necessary.
Fetal bovine serum [GEMINI 100–106] is added to HBS solution to a final FBS concentration of 20%. Store at 4°C for up to 3 months.
Dissolve 50mg each of collagenase A [Roche 11088785103] and collagenase B [Roche 11088823103] in 5ml of flow cytometry buffer. Store at 4°C for up to 4 weeks. Prior to use, supplement an aliquot of the required volume with DNase [Calbiochem 260913] to a final concentration of 10µg/mL.
The self-renewal and developmental potential of pluripotent stem cells make them an attractive source for rare cell populations that arise during embryonic development, such as the earliest organ-specific progenitor populations, from which all or a large subset of cell types of a particular organ are derived. It has been demonstrated that such a population exists for the mammalian heart and is marked by positive Nkx2.5, Isl1, and/or Flk-1 expression in mice (Wu et al., 2006, Moretti et al., 2006, Kattman et al., 2006) and KDR-1 or Isl1 expression in differentiated human ES cells (Yang et al., 2008, Bu et al., 2009). These progenitor cells give rise to cardiomyocytes, smooth muscle cells (Wu et al., 2006, Bu et al., 2009), and endothelial cells (Bu et al., 2009), the three primary cell types of the heart. Although cellular markers for isolation of cardiac progenitor cells from in vivo and in vitro contexts have now been identified, the process by which a pluripotent stem cell first becomes committed to a multipotent lineage progenitor remains largely unknown. Further inquiry into these processes holds vast potential for clinical translation, particularly in the areas of congenital heart disease and regenerative therapy for myocardial injury.
The developmental potential of ES or IPS cells is strongly affected by a number of different factors, the most important of which is their ability to remain uniformly undifferentiated prior to initiating differentiation. During propagation, the cells should always be maintained in fresh media and be passaged before reaching 80% confluency in the culture dish. As the passage number increases (>30), the quality of karyotype deteriorates and the differentiation capacity of ES and IPS cells declines. It is advisable to use lower-passage cells for experimentation. Cells that remain undifferentiated should exhibit small size and round shape with large nuclear-to-cytoplasmic ratio. When cultured on feeder MEF, they should aggregate into larger colonies with raised borders and refractile surfaces. They should be free of any contamination, including mycoplasma. Typical signs of poor quality cells include the appearance of large numbers of cellular fragments floating in the culture medium, the presence of vacuoles on cell surface or within each cell, and colonies with irregular borders and flattened cell morphology. If the culture media turns yellow or yellow-orange in color earlier than usual, a suspicion should be raised for the presence of mycoplasma contamination. It has been our experience that cells exhibiting these defects do not differentiate well and should be discarded.
The efficiency of differentiation into the cardiac lineage is also influenced by the particular lot of fetal bovine serum used to make differentiation medium. Several different lots should be tested, and the one that yields the highest percentage of eGFP+ cells at Days 7–10 of differentiation should be used exclusively for future experiments.
Finally, cell density at the outset of differentiation is also an important factor contributing to the yield of cardiac progenitors. For both EB and monolayer differentiation protocols, the stated number of cells per droplet or well, respectively, have been empirically determined to maximize the number of eGFP+ cells obtained. In adapting the protocols of this unit for ES or IPS lines carrying different transgenic cardiac markers, it may be necessary to adjust the cell seeding density for optimal differentiation.
The protocols outlined in this unit can be scaled up to yield large numbers (105 – 106) of Nkx2.5+ cardiac progenitors for a variety of applications directed towards investigating early cardiac development and the therapeutic potential of progenitor populations and their derivatives. Upon continued culture, Nkx2.5+ progenitor cells should spontaneously differentiate into beating cardiomyocytes and smooth muscle cells.