Stem Cell Know-How

Image: Courtesy of Gwenn-AEL Dnaet ©2002 National Academy of Sciences STEM CELL XENOGRAFT: Identification of human hepatocytes in livers from immune-deficient mice transplanted with human adult hematopoietic stem cells. Photomicrographs of NOD/SCID mouse liver sections from mice transplanted with purified human Lin-CD38-CD34-C1qRp+ cells isolated from umbilical cord blood, harvested 8-10 weeks post-transplant. Tissue sections were stained for HSA (hepatocyte-specific antigen) or c-met

Sep 2, 2002
Aileen Constans
Image: Courtesy of Gwenn-AEL Dnaet ©2002 National Academy of Sciences
 STEM CELL XENOGRAFT: Identification of human hepatocytes in livers from immune-deficient mice transplanted with human adult hematopoietic stem cells. Photomicrographs of NOD/SCID mouse liver sections from mice transplanted with purified human Lin-CD38-CD34-C1qRp+ cells isolated from umbilical cord blood, harvested 8-10 weeks post-transplant. Tissue sections were stained for HSA (hepatocyte-specific antigen) or c-met (hepatocyte growth factor receptor). (a, g) Human liver section positive controls. (b, h) noninjected NOD/SCID mouse liver negative controls.

Stem cells make for hot news. Debates over the ethics of using human embryonic stem cells in research have topped headlines, and current research into the plasticity of adult stem cells has raised hopes that these cells could be a suitable replacement for their embryonic counterparts in a number of therapeutic applications. But often overlooked is the fact that stem cells are tough to grow. They are much less forgiving than regular cell lines, and so greater attention must be paid to their culturing. As it happens, the tools and techniques used by stem cell researchers represent an interesting twist to conventional cell culture.

Embryonic stem cells (ESCs), are derived from the inner cell mass of the blastocyst, the developmental stage of the embryo prior to its implantation in the uterine wall. What distinguishes ESCs from other types of cells is their ability to differentiate into cells from all three embryonic germ layers, a quality referred to as pluripotency. Some researchers refer to ESCs as totipotent (the capability to differentiate into any of the cells of the body), but there is some debate over the appropriateness of this term, as a single ESC cannot by itself create a new embryo.1

ESCs are capable of self-renewal, the ability to divide indefinitely without differentiation. They can also maintain a full diploid karyotype, generate any tissue when introduced into an embryo, and colonize the germ lines of recipient embryos.1,2 The most common ESC lines used for research are mouse-derived, and the conditions required for the culture of these cells are well characterized.

WORKING WITH EMBRYONIC STEM CELLS To establish and maintain ESCs with a normal karyotype and the ability to fully differentiate into functional cells, scientists generally start by plating cells onto a feeder layer comprised of mouse embryonic fibroblasts (MEFs). In addition to supplying as-yet-unidentified support factors, the feeder layer supplies leukemia inhibitory factor (LIF), which prevents ESC differentiation. Once LIF is withdrawn from the culture, ESCs begin to differentiate.1 Scientists can bypass the feeder layer, thereby avoiding the pitfalls associated with feeder cells, by adding LIF to the growth medium of the mouse ESC culture. "Feeder cells aren't fun to work with," explains Mark Wight, technical services manager for Logan, Utah-based HyClone Laboratories, as they must be prevented from overcrowding the ESC colonies. Wight notes, however, that most people working with stem cells continue to use feeder layers to avoid changing their experimental protocols.

Human ESC culture is a bit more complicated, as the mere addition of LIF to the growth medium fails to produce successful growth and maintenance conditions. Growing human ESCs on an MEF feeder layer, however, presents several problems. "If you think about how you might want to scale [human ESC] production so that you could use them to differentiate into cardiomyocytes, or islet cells, or neurons for eventual transplant into a person, you realize you're going to need lots and lots of cells," says Jane Lebkowski, vice president for cell therapies at Geron Corp in Menlo Park, Calif. This makes using feeder layers impractical. Also, complete separation of mouse feeder cells from the human cells is difficult. "One of the things you want to avoid in providing a therapeutic cell to a person is having ... a xenograft, or nonhuman cell, going back into a patient," Lebkowski adds.

To address these problems, scientists at Geron developed a feeder-free human ESC growth protocol that employs a MEF-conditioned medium (or conditioned medium from other cell lines) combined with extracellular matrix proteins, which are required for adhesion of cells to the culture plate and for the binding of cellular growth factors.3

The composition of the growth medium is another variable to consider when growing embryonic stem cells. ESCs are generally grown in a rich culture medium that includes the reducing agent 2-mercaptoethanol and 10-20% fetal bovine serum (FBS).1 But the quality and composition of FBS can vary, and researchers must routinely screen sera prior to use for effects on cell morphology, plating efficiency, and toxicity. HyClone and other companies sell prescreened serum, but laboratories can also test sera in-house.

The final step in an ESC culture protocol is to test the cells for proper karyotype and absence of differentiation, and for the ability of a single cultured cell to colonize a recipient blastocyst and create a chimeric embryo. "We routinely test [ESCs] by karyotyping," says M. Celeste Simon, associate professor, department of cell and developmental biology, University of Pennsylvania. "[But] the ultimate test for totipotency is that you can actually make a mouse out of a single cell."

WORKING WITH HEMATOPOIETIC STEM CELLS Hematopoietic stem cells (HSCs) are present in blood and bone marrow and give rise to blood and immune system cells. They are interesting to researchers because of their ability to undergo differentiation and self-renewal; and, unlike any other type of stem cell, are currently used in the treatment of diseases such as cancer.2 But HSCs are relatively rare and are difficult to identify by morphology alone; they are thus frequently characterized by functional assay. Several methods exist to enrich HSCs,4 including fluorescence-activated cell sorting (FACS), immunomagnetic separation, and density-gradient centrifugation.

Because of its ease of use and speed relative to FACS, immunomagnetic separation--in which targeted cells are labeled with specific antibodies and magnetic beads--is the most popular enrichment method. The technique can be used with both positive- and negative-selection strategies.

Scientists use positive selection to obtain high purities of progenitor cells by targeting a single cell-surface antigen, such as CD34, on the desired cells. But Debra Sauve, product manager for Vancouver, British Columbia-based Stem Cell Technologies, says some researchers prefer a negative-selection approach, in which unwanted cells, such as lineage-committed cells, are targeted for depletion, leaving an enriched progenitor population.

The choice of selection method depends on the relative abundance of the target cells and the application for which the cells will be used. Researchers looking for rare cells generally prefer the positive-selection approach. "If you're looking for a very rare cell, you have to throw in so many more antibodies to mop up all of the other unwanted cells [with negative selection], so any contaminants contribute significantly to the end result," says Sauve. Investigators who worry that binding of antibody-tagged beads will interfere with further studies tend to prefer negative-selection methods.

An alternative approach is density- gradient centrifugation, which separates cells according their migration through a buoyant density medium such as Ficoll®. This method is used to separate the denser red blood cells and polymorphonuclear cells from the less dense mononuclear cells, including HSCs, lymphocytes, and monocytes.

To facilitate separation of the mononuclear cells, Stem Cell Technologies has developed RosetteSep reagents that combine immunoseparation with conventional density-gradient centrifugation. The RosetteSep technique uses a mixture of antibodies to crosslink red blood cells to other unwanted cells in the sample. This increases the density of the unwanted cells such that they pellet when centrifuged over Ficoll. According to Sauve, the procedure is rapid, easy to use, and ideal for the enrichment of abundant cell types or as a pre-enrichment step for HSCs and other rare cells prior to FACS.

Once the sample has been enriched for the cells of interest, it is ready for culture. Two types of HSC culturing methods exist--clonogenic assays and expansion, explains Cyndy Nauer, technical support manager, Stem Cell Technologies. The choice of approach depends on whether the goal is to fully characterize or simply increase the number of progenitors.

In a clonogenic, or colony, assay, cells are grown in methylcellulose, a semisolid culture medium. The gel-like medium allows single, proliferative HSCs to multiply, differentiate, and form individual colonies that can then be counted and characterized. Colony assays have been used for a wide variety of clinical and research applications.

For expansion, scientists culture cells in a base liquid medium containing bovine serum albumin (BSA), human insulin, human transferrin, and a reducing agent such as 2-mercaptoethanol. Cytokines, or specific growth factors, appropriate to the culture of a specific HSC, are also added. Stem Cell Technologies, for example, offers specially formulated cytokine cocktails to accompany StemSpan™ serum-free expansion medium used to expand a variety of HSCs.

The goal of an expansion approach, as its name suggests, is to increase the number of HSCs with minimal differentiation. Nauer explains that some level of differentiation will always occur with a liquid expansion. In fact, the use of umbilical cord HSCs in cell therapy has been limited by the inherent difficulty of expanding and maintaining them in an undifferentiated state. Although the total number of progenitor cells one can obtain from cord blood is quite low, says Nauer, if those cells could be expanded to higher numbers, they could potentially be used for transfusion into adult patients.

A solution to this problem may be on the horizon, as two recent, concurrently published articles demonstrate.5,6 Jennifer Antonchuk and colleagues at the British Columbia Cancer Agency, University of British Columbia, and University of Montreal, showed that mouse bone marrow HSCs can be expanded 1,000-fold when transduced with the transcription factor Hoxb4.5 Researchers in George Daley's lab at the Whitehead Institute for Biomedical Research injected HSCs expressing Hoxb4 into irradiated adult mice and showed that the donor cells differentiated into lymphoid and myeloid cells.6 These findings suggest that transduction of HSCs with Hoxb4 could serve as a more efficient method for growing the large quantities of HSCs necessary for therapeutic applications.

Aileen Constans (aconstans@the-scientist.com) is a contributing editor.

References
1. A.G. Smith, "Embryo-derived stem cells: of mice and men," Annual Reviews in Cell and Developmental Biology, 17:435-62, 2001.

2. National Institutes of Health, "Stem cells: scientific progress and future research directions," available online at www.nih.gov/news/stemcell/scireport.htm.

3. C. Xu et al., "Feeder-free growth of undifferentiated human embryonic stem cells," Nature Biotechnology, 19:971-4, 2001.

4. T.E. Thomas et al., "Purification of hematopoietic stem cells for further biological study," Methods: A Companion to Methods in Enzymology, 17:202-18, 1999.

5. J. Antonchuk et al., "Hoxb4-induced expansion of adult hematopoietic stem cells ex vivo," Cell, 109:39-45, April 5, 2002.

6. M. Kyba et al., "Hoxb4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors," Cell, 109:29-37, April 5, 2002.