Isolation and maintenance of stem cells pose several challenges due to the propensity for stem cells to differentiate into a specific cell type. Isolation procedures are continuously evolving and vary for different types of stem cells such as embryonic stem cells (ESCs), adult stem cells, and induced pluripotent stem cells (iPSCs). Part of the challenge of maintaining stem cells in culture is the requirement for specialized media, growth factors, and potentially feeder cells. This section provides an overview of the isolation and maintenance of different types of stem cells and provides resources for protocols and procurement of stem cells.
Related Topics: Stem Cell Research, Differentiation of Stem Cells, Transfection of Stem Cells, and Analysis of Stem Cells.
Traditionally, embryonic cells were grown on a layer of mouse embryonic fibroblast (MEF) feeder cells, which can be purchased from the American Type Culture Collection (ATCC), or isolated by retrieving embryos from a pregnant animal. To isolate MEF from an animal, the head, liver, intestines, heart, and all viscera are first removed, and the remaining tissue is minced and sheared. The resulting suspension is divided among T175 flasks and grown at 37°C with 5% CO2 for 3–4 days. Before they can be used as feeder cells, the MEF cells must be mitotically inactivated using either UV light or mitomycin-C chemical treatment. This prevents the MEF cells from overwhelming the slow-growing stem cells in the culture. The MEF cells can be frozen before or after inactivation and then used as the stock for either generating more feeder cells or for supporting ES cells. Standard plating density for MEF cells to support ES cells is 1.2 x 105 cells/cm2 or about 1.2 x 106 cells in a 35 mm dish.
Methods to culture ES cells without feeder cell layers have also been developed. This originally required coating tissue culture plates with a gelatinous protein mixture isolated from mouse tumor cells. There are now commercially available media formulations for maintaining stem cells in their original state.
Preparation of mitotically inactivated MEF cells for use as feeder cells.
Human embryonic stem cells are available through commercial vendors. Embryonic stem cells from other model organisms such as mouse and rat are also available.
Alternatively, cell lines can be established by culturing zona-free blastocysts or morulae embryos in a feeder layer for 3–4 days. The inner cell mass (ICM) that propagates is harvested and the tissue dissociated before being replated and expanded. Once expanded to a high enough density, the cells are verified by screening for the proper surface markers, a normal karyotype, and the ability to form a teratoma in an immune-compromised mouse.
Cultivation of embryonic stem cells.
Somatic stem cells are found in most major organs and tissues; however, the number of cells that can be isolated varies greatly depending upon the origin. For example, cardiac stem cells are quite rare, while hematopoietic stem cells occur in high enough numbers that they are routinely isolated and used in medical procedures.
Isolation of somatic stem cells is done either by an enrichment process or through a depletion process. Enrichment is commonly used for isolating cells originating from the hematopoietic system. The isolated cells are used for treating patients with a number of diseases including multiple myeloma and leukemia. In this case, apheresis is a common technique used to collect the appropriate cells from circulating blood cells. In a research environment or when cells are isolated from bone marrow, density gradient centrifugation or bead-based isolation methods may be used. Beads are coated with antibodies to surface markers found on the cells of interest. The cells expressing these markers bind to the beads allowing the other cells to be washed away. The bound cells are eluted from the beads and cultured under proper conditions.
In a depletion process, cells from the tissue of interest are plated in media that will allow growth of only the stem cell. This process is typically used for isolation of adherent cell types such as cardiomyocytes. In this case, cardiac cells are prepared from tissue biopsies and grown in media that select only for stem cells. The cells resulting from the culture procedure are verified for stemness by all the methods cited previously.
Sources of somatic stem cells in the human body.
Methods for creating iPS cells are continuously evolving (see stem cells overview for details). At the time of writing, the presence of a single transcription factor, Oct4, was enough to induce pluripotency in a certain cell type (Kim et al. 2009). The introduction of this factor can be done through a virus (Kim et al. 2009), electroporation, or the transcription factor itself. Protocols for doing these are identical to those used in standard molecular biology experiments.
Kim JB et al. (2009). Direct reprogramming of human neural stem cells by Oct4. Nature 461, 649–643. PMID: 19718018
Hogan B et al. (1994) Manipulating the Mouse Embryo, second edition (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press).
Robertson EJ (1987) Teratocarcinomas and Embryonic Stem Cells: A Practical Approach (New York, Oxford University Press).
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