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Stem Cells
Concise Review

Stem Cell Assays: Something Old, Something New, Something Borrowed

Ronald van Os, Leonie M. Kamminga, Gerald de Haan Department of Stem Cell Biology, University of Groningen, The Netherlands
Key Words. Stem cells Competitive repopulation Competitor Reconstitution Assay

Abstract
Numerous assays exist that measure the function of stem cells. In this article, we review in detail the history and future of existing stem cell assays. Hematopoietic stem cells (HSCs) are historically the most well studied, but new developments in stem cell research, including the claim of stem cell plasticity, have caused controversies related to technical issues, as well as to semantics. Stem cell research requires proper definitions, and utilization of stem cell assays, especially since research on non-HSCs that lack solid stem cell assays, is rapidly evolving. These emerging fields may benefit from what has been learned from HSC assays: most important, that the true potential of stem cells can only be assessed retrospectively. This also relates to new developments in HSC research, when limiting numbers of in vitromanipulated stem cells are transplanted. The most conflicting results arise when cells express stem cell characteristics in one assay but not in another. Should we adjust our definition of a stem cell? If so, when do we decide a claim of stem cell activity to be justified? We therefore recommend using multiple stem cell assays, preferably at least one in vivo assay. These assays should measure functionality of the putative stem cell population. Stem Cells 2004;22:11811190

Something Old: A Historical Perspective The Hematopoietic Hierarchy

Blood contains multiple distinct cell types such as erythrocytes, granulocytes, lymphocytes, monocytes, and platelets. The finite lifetime of these mature cells requires a well-organized system of replenishment and cell renewal. The concept of how HSCs and progenitors are organized has been under continuous reappraisal since the 1960s. Taken together, various assays exist to measure the frequency of hematopoietic stem cells (HSCs) or progenitors. These assays include in vitro clonogenic assays, in vitro phenotyping, and in vivo transplantation assays. Decades of research resulted in the discov-

ery of a primitive, pluripotent HSC population that has the special ability to continuously and long-term repopulate all blood lineages, myeloid and lymphoid, of an irradiated recipient after bone marrow transplantation (BMT). Thus, this stem cell has long-term repopulation ability (LTRA). This activity can only be adequately shown in chimera models in which it is possible to distinguish between donor and recipient stem cell pools after BMT [1]. Upon transplantation of a cell population into lethally irradiated recipients, two distinct features of the injected population can be determined. The transplanted cell population must provide radioprotection to rescue the lethally irradiated recipient from radiation-induced bone marrow aplasia, and it must be able

Correspondence: Ronald van Os, Ph.D., Department of Stem Cell Biology, University of Groningen, A. Deusinglaan 1, NL9713 AV Groningen, The Netherlands. Telephone: 31-50-363-2722; Fax: 31-50-363-7477; e-mail: r.p.van.os@ med.rug.nl Received April 26, 2004; accepted for publication July 30, 2004. AlphaMed Press 10665099/2004/$12.00/0 doi: 10.1634/stemcells.2004-0095

STEM CELLS 2004;22:11811190

www.StemCells.com

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Stem Cell Function and Assays

stem cell frequencies other than in vivo long-term engraftment studies; these systems have provided a basis for useful comparisons with, for example, LTRA radiosensitivity [12] and cytotoxicity [13]. Advances in flow cytometry have revealed important information on the phenotype of HSCs. Although multiple approaches have been pursued over the years, the general consensus is that stem cells lack lineage markers (antigens that identify mature granulocytes, macrophages, B and T lymphocytes and reticulocytes), but they do express Sca-1, as well as c-kit [14, 15]. Other researchers added rhodamine or used Hoechst to identify cells with abundant dye efflux capacity [1419]. Also, stem cells are considered to reside in the LinSca-1+Thy-1loc-kit+ population [2022]. In the last decade, a new method has been developed to exploit the feature of quiescent stem cells and to exclude Hoechst dye. The display of Hoechst fluorescence simultaneously at two emission wavelengths revealed a small and distinct subset of cells that had HSC characteristics [23]. This population has since been called the side population (SP), and subpopulations within this population (Tip-SP-CD34 cells) are claimed to be true stem cells since they exhibit an unexpectedly high homing efficiency [24]. Phenotyping for stem cell markers may therefore be considered a surrogate stem cell assay, although much debate still exists on the true phenotype of HSCs. Generally, in situations in which stem cells are isolated from normal, unperturbed mice, there is a good correlation between phenotype and in vivo reconstitution potential. The in vitro stem cell assays based on long-term bone marrow culture and phenotyping are useful to quantify stem cells in a cell population, but it remains unclear whether all these assays are a good predictor of stem cell activity under all conditions. In addition, several reports have shown that with human cells, the CAFC assay generated results that were different from those of the LTC-IC assay, which seem to be dependent on the stromal cell line used and the accessory cells [2528]. Furthermore, these assays lack measurement of in vivo homing capacity, which is an essential trait for HSCs. The gold standard for stem cell activity therefore remains in vivo repopulation, but the standard competitive repopulation assay may be underestimating the stem cell activity of certain putative stem cell populations.

to provide permanent long-term engraftment. Without a bone marrow transplant, lack of cells capable of rapidly reconstituting the hematopoietic system would lead to mortality. The repopulation assay became the gold standard to demonstrate the long-term repopulating ability of HSCs.

Hematopoietic Stem Cell Assays

The term chimera is derived from the mythological creature with a lions head, a goats body, and a serpents tail. In biology, a chimera is generally defined as an organism that has cell populations from genetically distinct individuals. In the field of BMT, a radiation chimera is used to describe an irradiated recipient whose hematopoietic system is partly or fully derived from recognizable donor bone marrow cells. Complete chimerism is found when all blood cells are descended from donor stem cells. In a mixed chimera, both donor and host contribute to hematopoiesis. The assays available to distinguish between donor and host cells in the treated recipient use either cytogenetic, immunological, or biochemical markers. The technique of monitoring chimerism in congenic mice enables assessment of short-term engraftment (originating from colony-forming unitspleen [CFU-S]like cells) and long-term engraftment (originating from LTRA cells) following ablation of composite host stem cells by radiation. However, in vivo HSC assays usually make use of congenic mouse strains in which immunological rejection plays only a minor role. Long-term bone marrow cultures showed that HSCs could be cultured on a pre-established stromal layer, thereby allowing growth of stem cells in close association with supporting stroma [2]. This method was modified to measure the frequencies of different hematopoietic cell subsets growing as cobblestone areaforming cells (CAFCs) underneath the stromal layer [35]. It was demonstrated that CAFC frequencies determined at various culture times showed good correlation with the different hematopoietic subsets as tested with other assays (CFU in culture, CFU-S on day 12 [CFU-S-12], and marrow-repopulating ability). The CAFC assay has also been used for determining the sensitivity of the different hematopoietic cell subsets for radiation [6], cytotoxic drugs [7, 8] and purging protocols [9]. An assay using a similar approach but a different endpoint is the long-term cultureinitiating cell (LTC-IC) assay. The stem cells are also grown on stromal layers, but the endpoint is the presence of progenitor cells, with colony-forming ability. This assay was initially developed for studying human stem cell growth in vitro [10] but was shown to be also useful for measuring murine stem cell frequencies and can even be altered to support growth of lymphomyeloid progenitors [11]. Until now, the CAFCs and the LTC-IC assays are the only testing systems that have the ability to reliably measure mouse primitive

Something New: New Developments in Stem Cell Assays Recent Developments in Competitive Repopulation Assays
The standard method of competitive repopulation uses a fixed number of freshly isolated, unseparated bone marrow cells as competitors. Several motives have arisen to adapt this

van Os, Kamminga, de Haan

standard method, mainly to increase the likelihood of detecting stem cell activity. The first of these modifications used bone marrow cells from serially transplanted mice that are enriched for short-term repopulating stem cells but have lost most of their long-term repopulating ability [29]. Such compromised competitors ensure survival of recipients while simultaneously providing selective pressure on the test population to be identified as stem cells with LTRA. More recently, to avoid two time-consuming serial transplantations, cells that lack markers present on long-term repopulating cells were transplanted as competitors. In two examples, small numbers of in vitroexpanded highly purified stem cells were transplanted into lethally irradiated recipients, along with competitor cells that lack LTRA, in this case Sca-1 cells [30, 31]. A similar method was used in the first study showing multilineage engraftment of a single, prospectively isolated, transplanted cell [32]. This study showed that within the highly purified LinSca-1+c-kit+ (LSK) population, cells lacking expression of CD34 are most potent in long-term repopulation. As competitors, the CD34+ population within the LSK population was used [32]. Another method to increase the probability of detecting stem cells is the use of W/Wv mice (officially designated as WBB6F1/J-KitW/KitW-v) as recipients. These mice have a mutation in the gene coding for the c-kit receptor, are mildly anemic, and have a severe stem cell defect, allowing engraftment of transplanted cells without conditioning of the recipient. In this model, the transplanted wild-type cells have a competitive advantage over genetically deficient host stem cells that serve as competitors. This model allows engraftment of single stem cells at near absolute efficiency [33]. However, also in normal recipients, efficient homing from a highly purified stem cell population (Tip-SP CD34LSK) was observed because more than 95% of animals transplanted with a single stem cell showed more than 1% engraftment. In this study, a low dose of normal bone marrow cells was used as competitors [24]. Recently, the potential of HSCs to differentiate into distinct tissues to test the concept of stem cell plasticity has been investigated. Single cells have been transplanted, along with a competitor cell population that allowed survival of the lethally irradiated recipients but that contained minimal contribution to long-term hematopoiesis. Two groups were able to show differentiation of a single SP cell (sorted on basis of Hoechst dye exclusion [34]) into hematopoietic cells, as well as muscle tissue [35, 36]. The use of this particular competitor cell population demonstrated that at a clonal level, some stem cells might contribute to regeneration of distinct tissues. All these studies aimed to detect rare stem cells have relied on a stringent, compromised, competitor cell popula-

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tion. One important reason for this stringent selection is that transplantation of a small number of stem cells present in the test population can easily be out-competed by a competitor cell population from normal, unseparated bone marrow and thus remain undetected. Another reason to use an alternative source of competitors is to increase the likelihood of detecting stem cells with a competitive disadvantage. However, recently there have been a number of studies showing an unexpected discrepancy between hematopoietic activity in a competition assay and hematopoietic activity under noncompetitive conditions, which will be described in more detail below.

Discrepancies between Competitive and Noncompetitive Transplantation Assays

In recent years, several models have shown a severe stem cell defect in competition assays but fairly normal reconstitution potential when HSCs are transplanted without competitors (Table 1). HSCs from mice deficient for both isoforms of STAT5 (STAT5a and STAT5b/), for example, exhibit a severe engraftment defect when they are transplanted with competitors [37, 38]. However, STAT5-deficient stem cells were able to repopulate lethally irradiated recipients when they were transplanted without competitors [38]. In addition, chimeric mice generated by transplanting STAT5-deficient cells were susceptible to engraftment of wild-type cells without any further conditioning [39]. STAT5 is a member of a family of transcription factors that are active in many cell types and which can be activated by numerous cytokine receptors, G-proteincoupled receptors acting through intrinsic receptor tyrosine kinases, as well as by nonreceptor tyrosine kinases. STAT5 is believed to be critical for anti-apoptosis and proliferation. Constitutively activated STAT5 has been found in tumor samples such as lymphomas and leukemia, and it leads to cell cycle progression and upregulation of anti-apoptotic proteins such as bcl-xL [40]. However, transgenic overexpression of bcl-2 was not sufficient to correct the stem cell competition defect in STAT5-deficient mice [41]. In summary, STAT5 is an example of a protein that in deficient stem cells negatively affects self-renewal and, consequently, competition after transplantation; however, the overexpression of STAT5 results in uncontrolled proliferation and thus indefinite selfrenewal. This suggests a relationship between self-renewal and stem cell competition. In addition, STAT5 may be considered a key factor in determining stem cell competition as a measure of self-renewal on one site and malignant behavior on the other site. Ikaros-deficient stem cells were also able to reconstitute lethally irradiated recipients, but they had a more than 30fold reduction in competitive repopulation units [42]. A third

1184 Table 1. Severe competition defects with no effect on reconstitution capability Cell source Stat5a/5b/ donors Ikaros donors HoxA9/ donors Donors treated with multiple cycles of chemotherapy Serially transplanted stem cells
a /

Stem Cell Function and Assays

Fold reduction in CRa >25-fold >30-fold 8-fold 8-fold (3 5-FU)

Reconstitution capabilityb +/ + + +

Reference Bunting et al. [38] Nichogiannopoulou et al. [42] Lawrence et al. [43] Noach et al. [49]

>50-fold

Kamminga et al. [50]

CR was performed using freshly isolated, unseparated bone marrow competitors. The reduction in repopulating units compared with wild-type or untreated cells (equal numbers) is given. b Reconstitution capability refers to the potential of transplanted cells to radioprotect a lethally irradiated recipient and to restore blood cell production. Abbreviation: CR, competitive repopulation.

example was presented at the Annual Meeting of the American Society of Hematology in 2003 and showed that stem cells from mice deficient for HoxA9 have a competition defect [43]. These cells exhibited an eight-times lower repopulating ability than wild-type stem cells, and it was suggested that the mutation resulted in a functional rather than a quantitative stem cell defect. Other models that show stem cell repopulation defects include W mutant mice [44], strain description Inpp5d (commonly called SHIP)deficient mice [45], and Fanconi Cdeficient mice [4648], but the repopulation defects are mild and may be related to reduced cytokine responses. Finally, two additional nongenetic models showing competitive disadvantage were developed in our own laboratory. In the first model, mice that were treated with three consecutive doses of 5-fluorouracil showed normal stem cell numbers and a normal homing ability, but upon transplantation in a competitive assay, stem cell functioning deteriorated over time. This indicated an in vivo competitive disadvantage compared with untreated stem cells [49]. The second model is shown in Figure 1, in which bone marrow cells serially transplanted for five times were tested in three distinct stem cell assays [50]. Both the in vitro CAFC assay and the reconstitution of lethally irradiated recipients showed detectable stem cell activity. However, when these cells were tested in a competitive repopulation assay, no stem cell function could be detected. Moreover, fresh bone marrow cells engrafted, without any further conditioning, in mice reconstituted 3 months earlier with cells that had been serially transplanted five times [50]. Thus, as summarized in Table 1, a competitive disadvantage does not necessarily mean that cells are incapable of in vivo reconstitution.

Stem Cell Genes and Stem Cell Self-Renewal

In the era of genome projects, molecular biological tools have helped determine the expression of genes specific for stem cells. By definition, stem cells are able to differentiate into multiple lineages and to self-renewthat is, maintain their own numbers. Stem cell self-renewal is tightly regulated, and the proper expression of stem cell genes is crucial to ensure self-renewal. It is not unlikely that competitive disadvantage is caused by deregulated self-renewal. Self-renewal genes are believed to be highly expressed in HSCs, and constitutive overexpression of these genes may be involved in leukemia. If these are correct, competitive disadvantage may be directly related to stem cell self-renewal. Therefore, it is conceivable that self-renewal genes may be directly or indirectly downregulated in stem cells with a competitive disadvantage, which would mean that altered expression of self-renewal genes causes cells to function improperly when compared with normal stem cells. The opposite may be very relevant for leukemia in which enhanced self-renewal is often accompanied by increased competitive advantage over normal cells. This would imply that (stem) cells in patients suffering from acute myelogenous leukemia have a competitive advantage over normal cells, which leads to an overgrowth of the malignant cells. Moreover, in patients undergoing reduced-intensity conditioning (incomplete host stem cell elimination) in the setting of an allogeneic stem cell transplant, the transplanted cells may benefit not only from the enhanced immune suppression but also from a possible competitive advantage over severely treated endogenous stem cells. Recently, several proteins have been identified that are believed to determine stem cell fate, including HoxB4, HoxA9, Bmi-1, FGF-1, Notch, and Wnt [30, 31, 43, 5156].

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Figure 1. Variation in outcome of stem cell function measurements. Mice were serially transplanted with 4 106 unfractionated BMCs every 4 months. After five serial transplantations, stem cell function was measured using three different assays. First, the in vitro CAFC assay revealed a frequency of primitive, late CAFC day 35 of 1 in 500,000. Second, in a reconstitution assay, these stem cells provided normal blood cell reconstitution after in vivo transplantation. Third, in a competitive repopulation assay, when competed with normal, untreated stem cells, no stem cell activity could be detected. Therefore, the results of measuring stem cell function depend on the applied assay. Abbreviations: BMC, bone marrow cell; CAFC, cobblestone areaforming cell; TBI, total boy irradiation.

Expression of these genes is now considered necessary for maintaining stem cell characteristics. Mice deficient in STAT5, Bmi-1, or HOXA9 were shown to have impaired stem cell function, but overexpression of these genes has been implicated in leukemia [38, 43, 53, 54, 5759]. This information supplies evidence for the hypothesis that competitive disadvantage and leukemic transformation are correlated with, respectively, downregulated and upregulated expression of stem cell self-renewal genes. It has been suggested that selfrenewal is a default pathway that is lost during differentiation [60]. However, additional mutations apart from mutations affecting self-renewal, which interfere with death and survival and with differentiation, may be required for leukemic transformation. However, a mutation in a single gene may also cause altered expression of additional genes. For example, HoxB4-deficient cells have been shown to have deregulated expression of neighboring Hox genes [61]. HSCs transduced with HoxB4 exhibited controlled expansion of stem cells in vivo [52, 62], whereas stem cells cultured ex vivo with exogenous HoxB4 protein retained their repopulation potential [63, 64] without inducing leukemia. This indicates the existence of a mechanism that limits the expansion of stem cell populations above a predetermined threshold. The cellular and molecular characterization of this mechanism will have important clinical implications since it is clearly perturbed in diseases such as leukemia and some forms of bone marrow failure. The identification of true self-renewing genes is still a major challenge. A self-renewing division results in two

daughter cells with properties identical to the initial cell. The daughter cells should therefore be capable of independently reconstituting a recipient. Technically, this requires in vitro self-renewal and separation of two (or more) daughter cells prior to transplantation. However, the optimal culture conditions to ensure in vitro self-renewal are not known. In addition, the cultured cells may have an altered cell cycle status and adhesion molecule expression, which may hamper their homing and thus their functioning after transplantation. The most successful attempts have used either bulk cultures of purified stem cell populations stimulated with classical hematopoietic growth factors, FGF-1 or HoxB4 that showed an increase in the number of cells with repopulating ability [55, 6366]. Detection of self-renewal divisions of single stem cells still remains a major challenge. Stimulation of single purified stem cells with stem cell factor and thrombopoietin showed in vitro divisions, but in vivo repopulation ability was reduced [67]. Recently, stimulation of stem cells with proteins such as Wnt3a showed an increase in repopulation efficiency, but no self-renewal at the single cell level was shown since clones derived from a single stem cell were not transplanted into separate recipients [31]. Alternative explanations for improved performance of Wnt3a-stimulated stem cells remain possible. In conclusion, in vitro stimulation of stem cells with the products of stem cellspecific genes may induce selfrenewal. Showing true self-renewal requires a stringent repopulation assay that increases the likelihood of the transplanted cells to generate sufficient offspring to be detected.

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Stem Cell Function and Assays

cells may allow detection of function after local transplantation, although quantification may be difficult. Although several examples for other tissues (muscle and liver) have shown functional improvement of tissue function following stem cell transplantation [79, 8285], the tissue-regeneration model that resembles the hematopoietic system most is probably the testicular system. To detect stem cell activity from cells isolated from the testis, a putative stem cell population was isolated and transplanted in the testis of W/Wv-recipient mice. This mouse strain lacks functional spermatogenesis, and by using stem cells from a transgenic mouse carrying the lacZ gene, the progeny of transplanted stem cells can be visualized [86, 87]. This has led to further phenotypical characterization of testicular stem cells [88]. In summary, many tissues lack functional in vivo assays to detect stem cell activity and, therefore, the development of additional models, both in vivo and in vitro, is vital in future studies on tissue stem cells, and plasticity of stem cells in particular. In addition, researchers should be aware of whether stem cell competition (with co-transplanted or endogenous stem cell populations) has a role in detection of stem cell activity and whether alternative (in vitro) stem cell assays are comparable under all conditions.

In the classical competitive repopulation assays, this may not be achieved.

Something Borrowed: Assays to Determine Stem Cell Function in Nonhematopoietic Tissues

Evidence has accumulated on the potential of bone marrowderived stem cell subsets to contribute to nonhematopoietic tissues, a property termed plasticity or transdifferentiation. We do not discuss here the potential mechanisms (fusion or true transdifferentiation) that may explain these findings, and we refer to a recent review on these issues by Herzog et al. [68]. Rather, we focus on the assays used to claim stem cell plasticity. In vitro growth of adult stem cells has been achieved with HSCs, mesenchymal stem cells (MSCs), and neural stem cells (NSCs). MSCs can be grown in culture, retaining their capacity to differentiate into mesenchymal lineages such as adipocytes, chondrocytes, and osteoblasts [69]. NSCs obtained from fetal brain can be maintained in vitro for a long time, and differentiation can be induced to generate neurons, astrocytes, and oligodendrocytes in vitro [70, 71]. However, these assays need to be validated with in vivo endpoints. Decades of research in the field of HSC biology have clearly documented that in vivo stem cell activity can only be observed if the transplanted test population has a numerical or qualitative advantage over the resident endogenous cells. In the field of hematopoiesis, this requires elimination of host stem cells by, for instance, radiation. However, in other tissues (brain, muscle, and heart), efficient elimination of endogenous stem cells may not be feasible without significantly compromising tissue function. To their rescue, investigators may borrow from experience accumulated in research on HSCs. Some characteristics may be similar for stem cells from different tissues, allowing prospective identification of tissue-specific stem cells. The SP, for instance, was shown to exist in multiple tissues [72, 73], and either the Sca-1 or the ckit antigen (or both) may be present on the surface of multiple tissue-specific stem cell populations [7478]. A major shortcoming in testing these tissue-specific stem cells, however, is that we cannot measure in vivo function as easily as in the hematopoietic system because (competitive) transplantation is cumbersome or even impossible. Exceptions may be the liver, the central nervous system, and the testis. In the liver, regeneration can be induced by partial hepatectomy or lesions caused by chemicals (carbon tetrachloride [CCl4]) or gene mutations (fumarylacetoacetate hydrolase) [73, 79]. In any case, quantification of donor cell contribution may be hampered by cell fusion and regeneration from recipient mature hepatocytes [80, 81]. In the central nervous system, some diseases are characterized by nonfunctional cells, and replacement of these cells by proper stem

Final Remarks
Historically, HSCs have been defined by two independent characteristics. First, they are multipotentialthat is, individual stem cells can produce all of the different cell types found in the blood. Second, true stem cells are self-renewingthat is, during the proliferation of stem cells, differentiation ought to be balanced by the maintenance of the stem cell pool. In vivo reconstitution and multilineage potential of long-term repopulating stem cells can only be assessed retrospectively in a wide variety of assays, most of which are reviewed in this article. Under normal steady-state conditions, the relationship between these assays, ranging from phenotype as determined by flow cytometry, in vitro clonogenic activity, to in vivo function, is generally good. Thus, all repopulating ability in normal bone marrow resides in the LinSca-1+Thy-1loc-kit+ [2022] or LinSca-1+c-kit+Rholo [15] or HoechstloRholo [17, 18] or SP [23, 24] population. However, in perturbed hematopoiesis, such as drug treatment, growth factor treatment, serial transplantation, and genetic alterations, the functional correlations between the various assays is often lost (Table 1). Therefore, a cell may express certain stem cell characteristics in one assay but not in another. Table 2 provides a summary of the different stem cell assays that we have discussed, with their advantages and disadvantages. It is obvious that the ideal stem cell assay allowing both detection and quantification of stem cell activity at all times does not exist yet and may never be developed. Alterations of standard repopulation assays using stem cell

van Os, Kamminga, de Haan

Table 2. Hematopoietic stem cell assays and their advantages and disadvantages Measures proliferative potential No Measures in vivo reconstitution capability No

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Assay Phenotyping (flow cytometry)

Advantages Fast

Disadvantages No function; phenotype may change as a result of experimental condition; not all strains of mice express all stem cell markers Labor intensive; read-out may change according to experimental condition

In vitro clonogenic growth (CAFC, LTC-IC)

Yes

No

Semi-fast, in vitro surrogate; allows quantification of different subsets in a single assay; does not require competitive advantage; can be used in any mouse strain Clinically most relevant; stringent assay not dependent on competition Quantification possible relative to normal; unperturbed stem cells; read-out may improve when alternative competitors are used

Intravenous transplantation (no competitors) Intravenous transplantation in a competition assay

Yes

Yes

Slow; no quantification possible; if no short-term repopulating cells are present, recipients die Slow; cells with competitive disadvantage are false negative; quantification is only possible when normal stem cells are used as competitors; can only be done in certain mouse strains

Yes

Yes, with limitations

Abbreviations: CAFC, cobblestone areaforming cell assay; LTC-IC, long-term culture-initiating cell assay.

depleted competitors may enhance detection of stem cells, for instance, when low numbers of cells are transplanted. This assay, although not yet standard, requires both in vivo differentiation and self-renewal of the transplanted cells, and it may thus be considered to measure true stem cell function. Stemness may not be a property per se, but the assay in which a cell is tested determines how much of its stem cell potential will be revealed. Thus, who and what will decide whether a claim of stem cell activity is justified? This is not only an issue of fundamental scientific debate but also a practical problem in accepting or rejecting manuscripts that claim stem cell activity on the basis of a certain stem cell assay considered inappropriate by a reviewer. Here we propose that, whenever feasible, two independent assays should be used, including, preferably, at least one in vivo assay that

measures long-term function of stem cells. This strategy may be the best safeguard against premature conclusions establishing scientific dogmas that take years to correct. This conclusion relates particularly to nonhematopoietic tissues for which solid in vivo stem cell assays have not yet been developed. The scientific world eagerly awaits new assays to gain insight into the processes that are required for stem cell function and stem cell plasticity.

Acknowledgments
This work was supported by grants form the Dutch Cancer Society (NKB 2000-2182), the Dutch Organization for Scientific Research (NWO) (grant number 901-08-339), and the National Institutes of Health (USA) grant number R01HL073710-01.

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Stem Cell Function and Assays

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