Course Design and Methods

Laboratory instruction

The goal of the laboratory instruction of the human embryonic stem cell culture training course is to give students hands-on experience in techniques used for human embryonic stem cell and neural stem cell culture. We will accept applicants only if they have documented previous experience in culturing embryonic stem cells (ES), other stem cells, and/or primary tissue cultures (see Trainee Selection, below). It will generally not be acceptable if applicants have experience only with established cell lines such as 3T3 or 293 cells.

The content of the laboratory exercises is given below. This will also be the order of the chapters in the course manual containing specific protocols that will be provided to each student. But because of time constraints, the temporal organization of the laboratories will not be as outlined below. For example, to expand human embryonic stem cells to the stage that they can be used for embryoid body formation takes several weeks, and differentiation after the embryoid body stage takes eight days.

To assure that the students will always have the appropriate cultures for each laboratory, the instructors will prepare the cultures in advance. This approach, which we have borrowed from Julia Child, will allow us to condense the time required so that all of the techniques can be experienced (Flow Chart).

The day-to-day schedule of laboratory sessions is provided in the Course Schedule. The laboratory training will aim to achieve expertise in four general areas:

1.A. Basic tissue culture techniques

In order to accommodate students with varying experience, there will be a half-day of instruction in basic tissue culture methods on the first day of the course. Previous experience with human material will not be required, and regardless of prior experience, the students will receive instruction about safely handling human cells and tissues. This will be followed with an overview of GLP (good laboratory practice) procedures, including appropriate dress, techniques, emergency procedures, and record keeping. Students will also learn methods of detecting and avoiding bacterial or fungal contamination and procedures to prevent cross-contamination of cultures so that different cell lines do not get mixed.

1.B. Human stem cell-specific methods

The human embryonic stem cell line to be used for the course has been described in detail in publications(Amit et al., 2000;Carpenter et al., 2001;Lebkowski et al., 2001;Xu et al., 2001;Thomson et al., 1998). Fresh or frozen-thawed donated human embryos produced by IVF were cultured to the blastocyst stage in G1.2 and G2.2 medium were subjected to embryonic stem cell isolation, as described for rhesus monkey embryonic stem cells. The inner cell masses were isolated by immunosurgery, with a rabbit antiserum to BeWO cells, and plated on irradiated (35 grays gamma irradiation) mouse embryonic fibroblasts. Culture medium consisted of 80% Dulbecco's modfied Eagle's medium (no pyruvate, high glucose formulation; Gibco-BRL) supplemented with 20% fetal bovine serum (Hyclone), 1 mM glutamine, 0.1 mM b-mercaptoethanol (Sigma), and 1% nonessential amino acid stock (Gibco-BRL). After 9 to 15 days, inner cell mass-derived out-growths were dissociated into clumps either by exposure to Ca⁺²/Mg⁺² -free phosphate-buffered saline with 1 mM EDTA (cell line H1), by exposure to dispase (10 mg/ml; Sigma; cell line H7), or by mechanical dissociation with a micropipette (cell line H9) and replated on irradiated mouse embryonic fibroblasts in fresh medium. Individual colonies with a uniform undifferentiated morphology were individually selected by micropipette, mechanically dissociated into clumps, and replated. Once established and expanded, cultures were passaged by exposure to type IV collagenase (1 mg/ml; Gibco-BRL) or by selection of individual colonies by micropipette. Clump sizes of about 50 to 100 cells were optimal. Cell lines were initially karyotyped at passages 2 to 7.

The human neural stem cell line SC23 (Figure 5) was derived from the frontoparietal cortex of a 25-week premature infant after obtaining parental consent. The mother tested negative for HIV, hepatitis B, VDRL, and HSV; the infant tested negative for bacterial pathogens. Tissues were digested in an enzymatic cocktail consisting of Dispase, DNase, and papain. After washing, the crude digest was plated onto fibronectin-coated Petri dishes in primary medium composed of DMEM/F12, 10% FBS, 20 ng/mL each human recombinant bFGF, EGF, and PDGF-AB, and 10% BIT9500 supplemented with glutamine, gentamicin, streptomycin, penicillin, amphotericin and ciprofloxacin. On each of the next ten days 50% of the medium was removed and replaced with identical medium less the FBS. As each culture neared confluence it was gently lifted with trypsin and passaged into no more than twice the surface area from which it was lifted. Karyotyping of this line revealed a normal 46XY chromosomal complement. Immunocytochemistry revealed extensive nestin, O4, and doublecortin staining prior to differentiation and extensive Tuj1, GFAP, and NeuN staining after differentiation. FACS showed that the cells express varying levels of CD34, CD133, CD9, CD81, CD24, CD54, AND CD56.

Preparation of medium

Stem cells require very specific media, and it is used in small quantities. Recipes (example given below) will be provided, and the faculty will discuss the composition of stem cell tissue culture media, and the role of the components in cell maintenance or differentiation. The faculty will demonstrate the preparation of sterile media.

Preparation of feeder layers and substrata

Human embryonic stem cells are usually cultured on layers of mitotically inactivated mouse embrynic fibroblasts (MEF). Since MEFs and an alternative cell type (SNL cell line) are available commercially, the students will not be trained to produce primary cultures. The students will be instructed in all of the subsequent steps: passaging the fibroblasts, inactivating them with mitomycin C, counting them to determine the appropriate density, plating the cells, storage of plates, and monitoring the effectiveness of the inactivation. Special attention will be paid to to the consequences of inadequate inactivation; the most common mistake made by novices is to fail to inactivate all of the cells, resulting in contamination of embryonic stem cultures with rapidly dividing mouse fibroblasts. Students will also be instructed in techniques for growing the embryonic stem cells without fibroblast feeder layers(Xu et al., 2001). As shown in Figure 6, the embryonic stem cells are cultured on an extracellular matrix (Matrigel) in medium that has been conditioned by feeder layers. Students will learn how to coat plates with Matrigel and with fibronectin (for the neural stem cells), and how to harvest conditioned medium.

Passaging cells

Human embryonic stem cells are routinely passaged by manual dissociation. Unlike mouse embryonic stem cells, the human cells cannot be enzymatically dispersed into single cells without causing unacceptable levels of death and differentiation. When the colonies reach 1-to-2 mm diameter, they are cut into 5 to 10 pieces with a pulled glass pipette and the pieces treated briefly with dispase (10/mg ml in embryonic stem medium). Colony pieces are then washed in PBS and re-plated on new fibroblast feeder layers. Normally this is done about once a week, but students will receive cultures previously prepared by course instructors. Before receiving their cultures, students will learn how to align the inverted phase microscope and to take photomicrographs. Before they dissect the colonies, students will learn to recognize embryonic stem colonies based on their morphology and will photograph them. The human embryonic stem cells have a typical morphology that differs from mouse embryonic stem cells and it takes practice to reliably recognize them. The students will be trained in the preparation of glass micropipettes, microdissection, and transfer of tiny fragments of colonies.

Neural stem cells are passaged by gentle trypsinization and replating at high density on fibronectin-coated dishes. Students will photograph the cells and passage them in preparation for characterization.

Freezing and thawing techniques

The most reliable method for freezing human embryonic stem cells is a vitrification technique(Reubinoff et al., 2001). Colony pieces are equilibrated in a solution containing 0.3 M sucrose, 20% DMSO and 20% ethylene glycol in embryonic stem medium, placed in a small volume in a plastic freezing straw, then flash frozen in liquid nitrogen. This is a far more unforgiving technique than other freezing methods. Even a brief delay in this procedure compromises the viability of the cells, so the students will practice the procedure several times without using cells before attempting to freeze human embryonic stem colonies. The students will then thaw the cells shortly after freezing them, replate them on feeder cells, and examine them the next day to determine whether the cells survive and remain undifferentiated.

Neural stem cells are cryopreserved by more conventional methods. They will be trypsinized, rinsed, and frozen slowly (surrounded by isopropanol in a controlled-rate freezing container) in a medium containing BSA, insulin, transferrin and 10% DMSO. After quickly thawing, the cells will be replated on fibronectin-coated dishes and examined the next day to detemine how well they survived.

1.C. Characterization of undifferentiated cells

In their undifferentiated state, embryonic stem cells are commonly characterized by high alkaline phosphatase activity (a trait first noted in primordial germ cells), and immunohistochemical detection of the cell surface epitopes SSEA-3, SSEA-4, Tra-1-60, Tra-1-81, and the transcription factor Oct 4. Neural stem cells are characterized by expression of CD133 and nestin. The laboratory on cell characterization will focus on the use of the markers shown in Table 2. The markers were chosen to represent the most commonly used assays for embryonic stem cells and neural stem cells.

Immunohistochemistry

Students will use an antibody to the SSEA-4 epitope (Chemicon) that is highly expressed on undifferentiated cells , and an antibody to nestin (Chemicon), which is a marker for neural cell precursors (Figure 7). embryonic stem cells and neural stem cells will be cultured on glass slides (LabTek) and fixed in paraformaldehyde by the instructors before the course begins. The students will incubate the slides with primary antibody (dilution will be determined by instructors in advance), which will be detected using fluorescent secondary antibodies. This exercise will give the students the opportunity to use a confocal microscope to detect fluorescent cells.

Alkaline phosphatase activity

Alkaline phosphatase activity will be detected in paraformaldehyde-fixed cultures using a reagent kit designed for detection of AP-labeled antibodies (Vector Labs). For this purpose cells will be cultured in organ culture dishes so that the whole culture will have good optical properties.

RT-PCR

The students will perform RT-PCR for detection of Oct 4 transcripts using the Oct-4 XpressPack™ (Chemicon) and nestin transcripts (Figure 8) in human embryonic stem cells and neural stem cells using published primer sequences. Transcript for a "housekeeping" protein (G3PD or b -actin) will be used as a control. RNA will be prepared with the QIAGEN RNeasy kit (QIAGEN).

Flow cytometric analysis

The course will include the use of flow cytometry as a means of evaluating levels of surface marker expression within a cell population. Flow cytometry will be performed with the assistance of the FACS facility at CHOC. The students will stain cells in suspension with antibodies to Tra-1-81, CD133, CD24, and CD34. See Table 2 and Figure 9 for expected results.

Cells for flow cytometry are resuspended in Ca²⁺/Mg²⁺-free PBS containing 0.02% sodium azide and 1% human albumin and divided into 100 m l aliquots containing approximately 5 x 10⁵ cells to which mAbs or isotype controls are added. Cells are incubated in the dark for 20 minutes and then washed with PBS. When the mAb is unconjugated, a secondary antibody (FITC goat anti-mouse or PE-conjugated sheep anti-mouse) is added and the sample allowed to incubate in the dark for an additional 15 minutes and again washed. At the completion of labeling, washed cells are resuspended in 200 m l of PBS containing 7-amino Actinomycin D (1 m g/ml). The cells will be sorted by FACS facility personnel. The students will be taught the principles of flow cytometric analysis and learn how to interpret the resulting data.

Karyotype

Time will not permit the students to culture cells to perform karyotype. Instead, the students will learn how to count and identify human chromosomes using slides prepared in advance by the instructors (Figure 10). The need for routine karyotyping will be emphasized, and the methods will be included in the manual.

Table 2. Markers for analysis of stem cells

1.D. Differentiation and analysis

Induction of differentiation

For differentiation of embryonic stem cells, embryoid bodies will be formed by culturing fragments of embryonic stem cell colonies using a modification of the hanging drop method. Individual colony fragments are placed in a small volume of differentiation medium in a well of a 96-well plate that has non-adherent U-shaped wells. After about 5 days the aggregates are transferred to glass slides, allowed to attach, and then fixed for immunohistochemical staining (Figure 10). Cells are induced to differentiate by removal of growth factors and addition of retinoic acid(Loring et al., 2001;McDonald, 2001;Nat et al., 2001;Reubinoff et al., 2000;Schuldiner et al., 2001;Zhang et al., 2001).

Differentiation of neural stem cells is much simpler. Although the cells can be dissociated and reaggregated to induce differentiation, the cells differentiate well even when left in monolayer culture. This will allow the students to perform FACS analysis on the differentiated neural stem cells and to easily visualize the cells with confocal microscopy (Figure 12).

Characterization of differentiated cells

Since bulk culture methods have not yet been developed for human embryonic stem cells, we do not expect to be able to provide enough differentiated cells for FACS analysis. We will have sufficient neural stem cells, however, so we will provide them to the students for use in their FACS analysis. The remainder of the analysis will be done by immunocytochemistry and confocal microscopy. The cells will be analyzed for the markers as indicated in Table 2. Three of the markers, beta-tubulin, GFAP, and O4, are expressed by neurons, astrocytes, and oligodendroglia, respectively, and are expected to be present in both of the cell lines after differentiation.