Stem Cell Assays: Something Old, Something New, Something Borrowed
http://www.100md.com
《干细胞学杂志》
Department of Stem Cell Biology, University of Groningen, The Netherlands
Key Words. Stem cells ? Competitive repopulation ? Competitor ? Reconstitution ? Assay
Correspondence: Ronald van Os, Ph.D., Department of Stem Cell Biology, University of Groningen, A. Deusinglaan 1, NL-9713 AV Groningen, The Netherlands. Telephone: 31-50-363-2722; Fax: 31-50-363-7477; e-mail: r.p.van.os@med.rug.nl
ABSTRACT
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 discovery 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 . 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 radio-protection to rescue the lethally irradiated recipient from radiation-induced bone marrow aplasia, and it must be able 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 lion’s head, a goat’s body, and a serpent’s 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 unit–spleen –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 . This method was modified to measure the frequencies of different hematopoietic cell subsets growing as cobblestone area–forming cells (CAFCs) underneath the stromal layer . 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 , and marrow-repopulating ability). The CAFC assay has also been used for determining the sensitivity of the different hematopoietic cell subsets for radiation , cytotoxic drugs and purging protocols . An assay using a similar approach but a different endpoint is the long-term culture-initiating 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 but was shown to be also useful for measuring murine stem cell frequencies and can even be altered to support growth of lymphomyeloid progenitors . Until now, the CAFCs and the LTC-IC assays are the only testing systems that have the ability to reliably measure mouse primitive 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 and cytotoxicity .
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 . Other researchers added rhodamine or used Hoechst to identify cells with abundant dye efflux capacity . Also, stem cells are considered to reside in the Lin–Sca-1+Thy-1loc-kit+ population . 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 . 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 . 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 . 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.
SOMETHING NEW: NEW DEVELOPMENTS IN STEM CELL ASSAYS
Evidence has accumulated on the potential of bone marrow–derived 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. . 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 . 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 . 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 , and either the Sca-1 or the c-kit antigen (or both) may be present on the surface of multiple tissue-specific stem cell populations .
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 ) or gene mutations (fumarylacetoacetate hydrolase) . In any case, quantification of donor cell contribution may be hampered by cell fusion and regeneration from recipient mature hepatocytes . In the central nervous system, some diseases are characterized by nonfunctional cells, and replacement of these cells by proper stem 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 , 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 . This has led to further phenotypical characterization of testicular stem cells .
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.
FINAL REMARKS
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 R01-HL073710-01.
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Key Words. Stem cells ? Competitive repopulation ? Competitor ? Reconstitution ? Assay
Correspondence: Ronald van Os, Ph.D., Department of Stem Cell Biology, University of Groningen, A. Deusinglaan 1, NL-9713 AV Groningen, The Netherlands. Telephone: 31-50-363-2722; Fax: 31-50-363-7477; e-mail: r.p.van.os@med.rug.nl
ABSTRACT
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 discovery 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 . 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 radio-protection to rescue the lethally irradiated recipient from radiation-induced bone marrow aplasia, and it must be able 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 lion’s head, a goat’s body, and a serpent’s 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 unit–spleen –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 . This method was modified to measure the frequencies of different hematopoietic cell subsets growing as cobblestone area–forming cells (CAFCs) underneath the stromal layer . 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 , and marrow-repopulating ability). The CAFC assay has also been used for determining the sensitivity of the different hematopoietic cell subsets for radiation , cytotoxic drugs and purging protocols . An assay using a similar approach but a different endpoint is the long-term culture-initiating 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 but was shown to be also useful for measuring murine stem cell frequencies and can even be altered to support growth of lymphomyeloid progenitors . Until now, the CAFCs and the LTC-IC assays are the only testing systems that have the ability to reliably measure mouse primitive 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 and cytotoxicity .
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 . Other researchers added rhodamine or used Hoechst to identify cells with abundant dye efflux capacity . Also, stem cells are considered to reside in the Lin–Sca-1+Thy-1loc-kit+ population . 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 . 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 . 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 . 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.
SOMETHING NEW: NEW DEVELOPMENTS IN STEM CELL ASSAYS
Evidence has accumulated on the potential of bone marrow–derived 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. . 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 . 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 . 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 , and either the Sca-1 or the c-kit antigen (or both) may be present on the surface of multiple tissue-specific stem cell populations .
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 ) or gene mutations (fumarylacetoacetate hydrolase) . In any case, quantification of donor cell contribution may be hampered by cell fusion and regeneration from recipient mature hepatocytes . In the central nervous system, some diseases are characterized by nonfunctional cells, and replacement of these cells by proper stem 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 , 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 . This has led to further phenotypical characterization of testicular stem cells .
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.
FINAL REMARKS
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 R01-HL073710-01.
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