A Side Order of Stem Cells: The SP Phenotype - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:14 |只看该作者 |倒序浏览 |打印

作者：Grant A. Challen, Melissa H. Little作者单位：Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia
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      【摘要】/ }' b( s" l  q9 H
   A defining property of murine hematopoietic stem cells (HSCs) is low fluorescence after staining with Hoechst 33342 and Rhodamine 123. These dyes have proven to be remarkably powerful tools in the purification and characterization of HSCs when used alone or in combination with antibodies directed against stem cell epitopes. Hoechst low cells are described as side population (SP) cells by virtue of their typical profiles in Hoechst red versus Hoechst blue bivariate fluorescent-activated cell sorting dot plots. Recently, excitement has been generated by the findings that putative stem cells from solid tissues may also possess this SP phenotype. SP cells have now been isolated from a wide variety of mammalian tissues based on this same dye efflux phenomenon, and in many cases this cell population has been shown to contain apparently multipotent stem cells. What is yet to be clearly addressed is whether cell fusion accounts for this perceived SP multipotency. Indeed, if low fluorescence after Hoechst staining is a phenotype shared by hematopoietic and organ-specific stem cells, do all resident tissue SP cells have bone marrow origins or might the SP phenotype be a property common to all stem cells? Subject to further analysis, the SP phenotype may prove invaluable for the initial isolation of resident tissue stem cells in the absence of definitive cell-surface markers and may have broad-ranging applications in stem cell biology, from the purification of novel stem cell populations to the development of autologous stem cell therapies.

PURIFICATION OF STEM CELLS VIA HOECHST EFFLUX: THE SIDE POPULATION PHENOTYPE

The ability to purify potential stem/progenitor cells relies largely on the expression of appropriate cell-surface antigens by the cells in question, such that they can be immunostained with fluorescently conjugated antibodies and isolated by fluorescent-activated cell sorting (FACS). In general, stem cell purification relies on judicious combinations of cell-surface markers, which can serve as either positive or negative markers for stem cell activity, none of which are by themselves specifically expressed on the stem cell. Although a major advance in the murine hematopoietic stem cell field came with development of phenotypic strategies to physically purify transplantable activity from bone marrow .

Stem cell biology in general, but particularly in solid tissues, suffers from the lack of specific cell-surface markers that unambiguously label all stem cells and/or only stem cells. Although recent inroads have been made in this field, such as isolation of neural stem cells from mouse brains using a peanut agglutininlow/heat stable antigenlow profile , many organ systems (such as lung, pancreas, and kidney) still lack definitive markers for isolation of progenitor cells. The search for specific markers of resident tissue stem cells is a major theme in stem cell biology today. The Hoechst efflux phenomenon has proven to be a highly useful primary purification strategy for isolating potential stem/progenitor cells from various tissues in the absence of cell-surface markers. Cells with an SP phenotype have now been described in many solid tissues, including the skeletal muscle, lung, liver, heart, testis, kidney, skin, brain, and mammary gland.
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MOLECULAR DETERMINANTS OF THE SIDE POPULATION PHENOTYPE
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How and why do these cells efflux dyes? The supravital stain Hoechst 33342 stoichometrically binds to AT-rich regions of the minor groove of DNA .9 ?( C/ u: H) l! [. C) ?8 h$ L1 m
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Figure 1. Fluorescent-activated cell sorting profile of murine bone marrow after staining with Hoechst 33342. (A): The side population (SP) appears as the Hoechst low fraction capable of pumping out the dye and typically represents 0.05%¨C0.10% of viable cells from murine bone marrow. The non-SP cells that retain high levels of Hoechst staining are also referred to as main population cells. (B): The SP is ablated when verapamil is included in the Hoechst incubation. Verapamil blocks the activity of drug transport proteins, preventing them from effluxing the dye.

It was hypothesized that Mdr1a/1b and Bcrp1 may have overlapping function and be redundant in determining the SP phenotype in mice. As such, the loss of either transporter alone would not disrupt the SP phenotype. Mdr1a/1b¨C/¨C mice contain normal numbers of SP cells in bone marrow, demonstrating that expression of Mdr1a/1b is not required for the SP phenotype .' y& V3 a1 P) j

Functional assays have shown Bcrp1¨C/¨C mice have normal numbers of HSCs in the total bone marrow, indicating that HSCs were maintained in these mice, but with the loss of this membrane transporter, they were located outside the SP region . This would seem particularly relevant for stem cells regardless of their tissue of residence given their potential requirement for ongoing proliferation throughout the life of the organism.

MARKERS OF SIDE POPULATION CELLS FROM VARIOUS TISSUES/ a. k: f, o0 w  x; P; h2 D

If the ABC transporters Bcrp1 and Mdr1 are responsible for the SP phenotype, then is it possible to use just these markers to sort prospective stem cells from your tissue of interest? It would appear not since different tissue SP cells may use different transporters and some level of Bcrp1 expression is often detected in various non-SP cells . If this is the case, then what other markers are shared between SP cells from different tissues? Is an SP cell in the bone marrow the same cell in the muscle? Does the SP represent a homogenous population of stem/progenitor cells? These are all issues that need to be addressed when considering the SP in your tissue of interest." b  |8 T& {0 d2 L7 M
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As determined by immunophenotyping, SP cells from non-hematopoietic tissues such as skeletal muscle, mammary gland, and testis share some phenotypic features with bone marrow, such as expression of Sca-1 and absence of mature hematopoietic lineage markers (Table 1). However, the expression of common stem cell epitopes is very heterogeneous throughout SP cells from various tissues, and each organ seems to contain a phenotypically distinct SP in terms of cell surface profile. Moreover, in many cases there is a large degree of immunophenotypic variation between SP cells from the same organ. For example, approximately 75% of murine liver SP cells express CD45, although the cells with the highest efflux capacity found at the lower tip of the SP tail were enriched for CD45¨C cells . Phenotyping data from the two aforementioned tissues show the SP from these organs is a heterogeneous population that may encompass contributions from both hematopoietic and resident tissue cells to the total SP pool.

Table 1. Phenotypes of murine side population cells from various tissues

A general issue with all stem cell purification strategies is that whereas assayable activity can be greatly enriched, the purified cell populations are still heterogeneous. In most cases, it is difficult to separate the most primitive cell compartment from closely related progenitors. The same is true of SP cells, and the need to rely on complex in vivo assays suggests that it may not be possible to rigorously address the exact phenotype of the cells responsible for stem cell activity in any SP fraction. It should also be noted that in the literature there are often large discrepancies between SP abundances from the same tissue. This relates to the many variables involved in the preparation and staining for isolation of SP cells by FACS. Because efflux of a dye is a dynamic process, slight variations in tissue dissociation, cell counting, Hoechst concentration, staining time and temperature, and stringency in selection of SP cells by FACS gating can dramatically affect the viability, homogeneity, and apparent yield of SP cells . Hence, although dye efflux is effective in obtaining a population of cells containing progenitor potential, in most cases the SP is still quite heterogeneous and shows tremendous variation caused largely by different preparation and purification protocols. Thus, the terms side population cell and stem cell should not be used interchangeably. Assays to determine the relative enrichment of in vitro and in vivo stem cell capacity of sorted SP cells must be used to determine optimal conditions for isolation of SP cells for individual tissue. Each organ possesses a unique set of conditions, and SP protocols must be optimized accordingly. Ultimately, the highest enrichment of stem cell activity from the SP fraction of any tissue will be achieved by purification using various cell-surface markers in combination with dye efflux." l. R* S$ {4 z- ~& }! C( b4 k

ORIGINS OF SIDE POPULATION CELLS THROUGHOUT DEVELOPMENT
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There is accumulating evidence to suggest hematopoietic cells are recruited to tissues such as muscle .
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Alternatively, organ- and tissue-specific SP cells may arise solely as a consequence of the normal development of that specific organ but share the SP phenotype as a function of their inherent biological characteristics. A report where bone marrow from transgenic GFP  donors was transplanted into the jugular vein of newborn W54/WV mice (genetic defect of c-kit receptor allows donor HSCs to colonize host bone marrow without irradiation) showed 8 months after transplant that the recipient testis SP was similar in size to previous studies (1.2%) but GFP  cells were negligible (< 5%) . The GFP  cells seen in recipient kidney SP could have been circulating hematopoietic cells trapped in the renal vasculature, although the kidneys were perfused before staining.2 K$ B/ v9 `( j) X6 M

CELLULAR POTENTIAL OF SIDE POPULATION CELLS7 O6 `6 _5 ?- o6 }5 U- x

With respect to whether an SP cell in one tissue is the same as an SP cell in another, this can also be investigated in terms of their cellular potential. SP cells from a range of nonhematopoietic tissues have been shown to have hematopoietic potential in vitro (with varying degrees of potency) when cultured under hematopoietic colony-forming conditions . The in vivo models harnessed to investigate stem cell potential typically involve some type of tissue injury as a priming event for proliferative expansion of transplanted cells to regenerate the damaged organ or expression of tissue-specific genes by donor cells in mutant mice. The methods used to induce and assess engraftment of donor cells will be stated in each of the following examples.5 u( p: H" J1 A1 O, l4 i
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SP cells initially drew great interest in the stem cell community due to the potency of long-term repopulating HSCs contained in the murine bone marrow SP fraction. In addition to the ability to reconstitute the hematopoietic system, murine bone marrow SP cells have now been shown to have nonhematopoietic lineage potential in vivo. In lethally irradiated female mdx (dystrophin gene mutation) recipient mice injected with bone marrow SP cells of male origin, 4% of myofibers were dystrophin , with 10%¨C30% of these fibers containing Y-chromosome  donor nuclei, showing that bone marrow SP cells have myogenic potential in vivo .; d" G! {1 `+ G2 c: J5 ^- s; M

Reconstitution of the lymphohematopoietic system of lethally irradiated mice by limiting numbers of HSCs is the gold standard for in vivo stem cell assays, and at this point in time, direct stem cell activity from highly purified SP cells has only been demonstrated for those derived from bone marrow. Although the SP fraction from numerous tissues has now been associated with stem cell activity, this generally has not been demonstrated to the same degree of stringency as HSC activity from bone marrow SP cells. This again reinforces the point that SP cells cannot be directly equated with stem cells until proven so with limiting numbers in a functional assay. Nevertheless, isolation of SP cells from various tissues does seem to enrich for resident tissue stem/progenitor cells in many cases (Table 2). Muscle SP cells clearly have hematopoietic potential in vivo, although they seem to be less potent than bone marrow SP cells. This was shown via the analysis of the bone marrow of irradiated mdx females into which muscle SP cells from male donors were injected .( F! k1 B& {! _: h$ Y* q

Table 2. Stem cell activity of side population cells isolated from various mouse tissues
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Kidney SP cells also seem to have hematopoietic potential, as well as a capacity to become other mature cell types. Ten weeks after transplant of SP cells from GFP transgenic rat kidneys injected into irradiated recipients, approximately 0.03% of bone marrow cells of recipients expressed GFP, indicating donor cells had engrafted into host bone marrow and produced progeny (although their developmental stages and lineages were not analyzed). Donor cells were also localized in laminin  skeletal muscle fibers and albumin  hepatocytes. In the kidneys of transplanted rats, proximal tubules showed green fluorescence that was brighter than autofluorescence observed in wild-type rats and than fluorescence observed in distal tubules or collecting ducts of SP transplanted rats .. j5 w! y& K: z

Liver SP cells certainly have the ability to give rise to a variety of cell types within the liver. Liver SP cells from Rosa26 transgenic mice transplanted into livers of recipient mice treated with DDC for 10 days before and after transplant engrafted as mature hepatocytes and bile duct epithelium in host livers, showing the hepatic SP cells had local regenerative capacity .

Other solid-tissue SP cells do not show apparent hematopoietic potential but do show multipotency. Skin SP cells from male donor mice injected into female mdx mice generated dystrophin-expressing Y-chromosome  muscle fibers 3 months after transplant . No donor cells were found in the spleen, and donor nuclei were not associated with regions of mononuclear cell infiltration, suggesting that these were not circulating cells. Thus, in nonirradiated hosts, skin SP cells did not differentiate into hematopoietic cells and likely did not remain in circulation, although they infiltrated damaged muscles and took residence there./ H& D4 t- N. T
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To test the potential of the mammary SP, SP cells were isolated from the mammary glands of Rosa26 transgenic mice and transplanted into cleared fat pads of immunocompromised Rag-1¨C/¨C recipient mice in limiting numbers .+ c/ C9 B" i" [( s0 X' q8 o

In recipient mice treated with busulfan to destroy endogenous spermatogenesis, donor testis SP cells transplanted via the efferent ducts showed a 13-fold increase in colonization efficiency compared with transplanted main population cells .
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The variability in potential of SP from different tissues can be interpreted as not supporting a common bone marrow origin for these cells. It can also be interpreted as suggesting a wide degree of differentiative potential for such cells upon exposure to various inductive environments. It is also highly likely that the apparent variability from one source of SP cells to another again reflects the heterogeneity of SP isolation from organ to organ. Tissue-specific SP cells can only be regarded as stem cells by clearly demonstrating that a single cell can give rise to multiple differentiated cell types of their tissue of origin.
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What has not been examined in close scrutiny in many examples of SP transplantation is the concept that spontaneous cell fusion rather than transdifferentiation might be the mechanism by which the donor cells acquire unexpected phenotypes and supposedly cross lineage boundaries. In a recent study where whole bone marrow from genetically marked reporter mice was transplanted into lethally irradiated recipients, engraftment of donor cells into recipient organs occurred not through transdifferentiation of the transplanted cell but by fusion of host and donor cells . Not only has this result and the fusion phenomenon cast suspicion about the validity of previous transplantation studies claiming expression of muscle gene products lacking in recipient cells after stem cell transplantation, but it now casts doubt over any transplantation study where fusion and/or reprogramming of the recipient genome was not examined.% A. a1 s8 O5 q: r3 W* L4 A
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THERAPEUTIC OPTIONS USING THE SIDE POPULATION PHENOTYPE
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The great hope for stem cell research is the potential development of cellular therapies for the treatment of various human diseases. One of the most widely used animal models in stem cell research is the mdx mouse, a model of Duchenne¡¯s muscular dystrophy that has a mutation in the dystrophin gene. Previous therapeutic strategies aimed at restoring dystrophin expression by cell transplantation . This supports the hypothesis that disease-damaged muscle could attract muscle SP cells from the circulation. The fact that lentiviral vectors can efficiently transduce a putative muscle stem cell and those cells can then contribute to the repair of a damaged muscle has substantial implications for possible autologous cell therapy of muscular dystrophy. Similar strategies could possibly see analogous therapies developed for a wide range of human single gene disorders, such as cystic fibrosis, Huntington¡¯s disease, and sickle-cell anemia.

MULTI-DRUG RESISTANCE, SP CELLS, AND STEM CELL PROPERTIES
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SP cells efflux dyes, and this property seems to be useful in enriching for stem cells. Why would a stem cell efflux vital dyes at a greater rate than other cells? One hypothesis to explain this phenomenon is that the overexpression of the membrane transporters responsible for the dye efflux mechanism in SP cells provides a mechanism for long-term survival of progenitor populations via an enhanced ability to pump cytotoxic compounds out of the cell . Therefore, it seems that progenitor cells can use Bcrp1/ABCG2 to reduce intracellular heme/porphyrin accumulation and overexpression of this membrane transporter confers a strong survival advantage under hypoxic conditions.0 G3 h- b+ v( |9 J' J5 c  j$ p6 G/ `
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What should also be considered is the known function of proteins such as MDR1. Some tumors are inherently resistant to most chemotherapeutic drugs (intrinsic resistance), and many others exhibit broad-spectrum or multidrug resistance after several rounds of chemotherapy (acquired resistance). A major cause of intrinsic or acquired multidrug resistance in humans is increased expression of the large (170-kD) cell-surface P-glycoprotein encoded by the MDR1 gene . This growing body of evidence implicating MDR1 in the protection of cells against a diverse range of cell death stimuli makes a lot of sense in terms of a stem cell population. Increased MDR1 expression in SP cells may confer a strong resistance to apoptosis as well as enhanced protection from xenobiotic compounds and be a contributing factor in long-term survival of these cells.
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If MDR1 is at least partly responsible for the SP phenotype, does that suggest a strong relationship between cancer cells and stem cells? Solid tumors contain cells that are heterogeneous in phenotype and proliferative potential. Because these malignancies are considered clonal in origin, it has been suggested that cancer cells in general may undergo processes that are analogous to the self-renewal and multilineage differentiation of normal stem cells , as would be expected given that the ability to efflux vital dyes is the basis upon which the cells were sorted from these tumors. The argument that this proves that the SP is a component of tumors and hence tumors are related to stem cells is a circular one. The SP phenotype should not be considered as more than one marker that can be used for the enrichment of putative stem cell populations. However, the assessment of relative SP numbers in tumor types may give us more information regarding the etiology of that tumor type.
      【关键词】 Side population Hoechst Stem cell Phenotype Dye efflux, |) c, c" t% E9 h0 f  K& D; Z
   CONCLUSIONS( N: Y: e& D8 T+ Q* g% B5 q

There is still a great amount of uncertainty about the origin, phenotype, and potential of SP cells from various tissues. Clearly the Hoechst efflux phenomenon has proven to be an extremely powerful tool to obtain enriched HSC populations from murine bone marrow, and this seems to correlate with the identification of putative stem cells in several solid organs. However, the dye efflux phenomenon is an active biological process, and because these prospective stem cells are not being isolated by a definitive cell surface profile, in many cases the purity of the population being isolated is very heterogeneous. Indeed, there are likely to be greater technical variations in the accurate isolation and quantitation of SP cells than cells isolated by direct immunopurification due to the dynamic nature of the parameter being measured (dye efflux). The fact that the proteins involved in the process of vital dye efflux itself may be used in other circumstances should always be taken into consideration before concluding that a cell with this phenotype is a stem cell. Despite this, there is evidence indicating that the SP is a good place to start in the search for resident stem cells in a particular organ when the phenotype of the cells in question is not known.
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ACKNOWLEDGMENTS* _: |/ f9 H2 j6 t5 j9 G% B" Z6 M

The authors would like to thank Professor Margaret A. Goodell (Centre for Cell and Gene Therapy, Baylor College of Medicine, Houston) for critical reading of the manuscript. Grant Challen holds an Australian Postgraduate Award. Melissa Little is an NHMRC Principal Research Fellow.

DISCLOSURES

M.L. founded and acts as a Director on the Board of Nephrogenix Pty Ltd./ ~* {$ _" `; }6 W, \3 {# f# g
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