Gather Ye Stem Cells Where Ye May

Stem cells are increasingly finding uses in both basic and clinical research. While traditionally of interest to those studying the regulation of growth and differentiation, stem cells are now being used in some of the hottest areas of research--in the construction of transgenic animals and vehicles in gene therapy and transplantation studies. These applications all require the culturing of stem cells outside the animal for some period of time, a task that is particularly challenging. As is the case with primary cultures of any kind, sustaining the life of and in some cases expanding a population of stem cells requires special conditions, different from those required by established lines. And with stem cell cultures, an additional problem is maintaining the pluripotency of the cells and, when appropriate, starting the cells off on the pathway of differentiation. A variety of special media and reagents can be purchased for each step of the way. Supplier of Purified Cytokines Alexis American QUALEX American Research Products Bachem Bioscience Becton Dickinson Biospacific Inc BioSource International Calbiochem-Novabiochem Endogen Chemicon Fitzgerald Industries International Genzyme HARLAN Bioproducts Hemagen Diagnostics ICN Biomedicals IGEN International Immunotech INTERGEN® Life Technologies Oncogene Research Products PeproTech R&D Systems Research Diagnostics Roche Molecular Biochemicals Sigma US BIOLOGICAL Pluripotent hematopoietic stem cells, which are capable of self-renewal and reconstitution of the hematopoietic system, comprise a very small percentage of the cells found in the bone marrow (0.05%). The therapeutic value of these cells for patients undergoing aggressive chemotherapy or requiring transplants has provided some of the impetus for developing methods for maintaining and growing these cells in vitro. Key to endeavors of this type are cytokines, of which literally dozens have been isolated. Cytokines have been described that stimulate the differentiation of stem cells into various cell types, called colony stimulating factors (CSFs), so called because they are assayed by the formation of colonies in special media--granulocyte-CSF, macrophage-CSF, granulocyte-macrophage CSF, and multi-CSF, which induces colonies of a number of lineages. In addition, there are lineage-specific cytokines, erythorpoietin and thrombopoietin, some of which are already finding clinical applications. Cytokines with the ability to expand the undifferentiated stem cell have been described as well, such as stem cell factor (SCF) and Flt-3 ligand. Because many of these factors may be in serum, serum-free media have been designed specifically for the propagation of pluripotent hematopoietic stem cells in culture: Life Technologies offers StemPro-34 for human hematopoietic cells, Quality Biological, Inc. has QBSF®-60, and Stem Cell Technologies offers two lines of media, human and mouse, to be used without serum and with different combinations of cytokines. These same companies also offer methylcellulose-based media, which are used for assessing the colony-forming ability of the stem cells (indicative of the ability of the stem cell to give rise to different differentiated cell types), as well as prescreened batches of serum that support colony formation in vitro. Transgenic mice have become a popular system for determining the function of genes or for modeling diseases, and one method for creating these interesting freaks of nature requires culturing mouse embryonic stem cells (ES cells) in vitro (A. Gossler, et al., Proc. Natl. Acad. Sci. USA 83:9065-9, 1986). Derived from the inner cell masses of blastocysts, ES cells are pluripotent and, while in culture, can be engineered to carry mutations or to pick up new genetic information. Once the desired tranformants are identified, these cells can be reintroduced into blastocysts, and when the ES cells are integrated into embryos, chimeric mice are formed, derived in part from the host and in part from the introduced cells. Chimeras that have germ cells originating from ES cells can then be used to create pure strains carrying the mutations, and to propagate them into future generations. In addition, ES cells can differentiate in vitro into little structures called embryoid bodies that contain a variety of cell types (endothelial, neuronal, muscle and hematopoietic progenitors), providing an interesting model system for studying development. Maintaining pluripotent or "undifferentiated" ES cells in culture is critical when using them to produce transgenic mice. ES cells remain pluripotent either by co-culture with feeder cells -- typically mouse embryo fibroblasts that have been irradiated or chemically treated to prevent them from dividing -- or by using a hormone called leukemia inhibitory factor (LIF) (Smith et al., Nature, 336:688, 1988; Matsui et al., Nature, 353:750, 1991). A preparation of murine LIF for use in ES cell culture is sold by Life Technologies via an exclusive licensing arrangement with AMRAD. Human LIF, which shows some activity with mouse cells, can be purchased as well. In addition, for optimum growth, ES cell culture media typically requires supplementation with fairly high concentrations (15%) of animal sera, generally fetal bovine serum (FBS). This can be problematic since FBS contains factors that can promote differentiation of ES cells, and is inconsistent from lot to lot. Therefore, researchers either must test each new lot of FBS prior to use, or avail themselves of the pre-tested products available from several companies. These products, termed "pre-qualified" by some, which are for the express purpose of maintaining pluripotent ES cell lines, are available from the companies listed below. Lexicon Genetics, Inc (Woodland, Texas) also offers a Mouse Kit, which provides the cell lines and reagents required for the production of a transgenic mouse. The low passage cell line AB2.2-Prime ES, licensed from Baylor College of Medicine, is featured in its offerings. The company also produces a line of vectors, the pKO Scrambler Series™ which can be used with its pKO Selection Vectors, for the generation of targeting vectors. Genome Systems, Inc., (St. Louis, MI) in addition to offering ES cells, has a complete set of materials for generating transgenic mice, in its "Go Germline" series. The ES cells provided are matched to a 129 mouse BAC library so that isogenic targeting constructs are easily obtained. This cell line produces color coat chimeras in the host strain, facilitating the identification of chimeric animals, and has also been shown to give germline transmission of desired alleles. In addition, a G418 resistant (to survive transformant selection) feeder cell line, MEF, which has been tested to ensure maintenance of pluripotent ES cells, is available. High density ES colonies were grown on inactivated mouse embryo fibroblasts in KNOCKOUT™ D-MEM (Life Technologies) LIF (1000 U/ml ESGRO™), and either 15% KNOCKOUT ™ SR (panel A) or 15% FBS (panel B). Some colonies are indicated by arrows. Photographs courtesy of Life Technologies. For those not quite ready to commit their labs to the production of transgenic mice, Genome Systems has custom services for every step in the production of transgenic mice--from genomic clone isolation, and vector construction, to blastocyst aggregation and karyotyping. Primary cell lines that retain their differentiated properties are desirable for many experimental applications. However, not all cultures of primary cells are successful--in many cases, the cells simply do not grow beyond a certain point, in other cases, spontaneous transformation occurs, resulting often in the selection of a particular fast-growing variant (many times not even from the tissue of origin). At best, supplementation of basic culture media seems a necessity, although the requirements for each cell or tissue type can and do vary. However, there is relief for those wanting to set up a quick primary culture without the time and patience to optimize their media. A number of companies now offer specialized media for particular primary cell types-- from neurons to colon and nearly every organ in between. The final table details what is currently available for the culture of primary cell lines. The author can be reached at ldefrancesco@the-scientist.com.

Gather Ye Stem Cells Where Ye May

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