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Cloned 20 years ago, stem cell antigen-1 (Sca-1) is used extensively to enrich for murine hematopoietic stem cells. The realization that many different stem cell types share conserved biochemical pathways has led to a flood of recent research using Sca-1 as a candidate marker in the search for tissue-resident and cancer stem cells. Although surprisingly little is still known about its biochemical function, the generation and analysis of knockout mice has begun to shed light on the functions of Sca-1 in stem and progenitor cells, demonstrating that it is more than a convenient marker for stem cell biologists. This review summarizes the plethora of recent findings utilizing Sca-1 as a parenchymal stem cell marker and detailing its functional role in stem and progenitor cells and also attempts to explain the lingering mysteries surrounding its biochemical function and human ortholog.
Disclosure of potential conflicts of interest is found at the end of this article.
Stem cell antigen-1 (Sca-1) is an 18-kDa mouse glycosyl phosphatidylinositol-anchored cell surface protein (GPI-AP) of the Ly6 gene family. Originally identified as an antigen upregulated on activated lymphocytes more than 30 years ago , Sca-1, or lymphocyte activation protein-6A (Ly-6A), is encoded by two strain-specific alleles [2, , –5]. The murine Ly6 gene family encodes at least 18 highly homologous, cross-hybridizing genes closely linked on mouse chromosome 15, many of which demonstrate greater than 80% sequence similarity with Sca-1 [2, 6, –8]. These cysteine-rich GPI-APs contain two or three protruding fingers, based upon the structure of Ly6 superfamily member cobra toxin proteins , and are localized to lipid rafts of the plasma membrane . Consisting of saturated sphingolipids and cholesterol, lipid rafts play critical roles in cell signaling by excluding or concentrating key signaling molecules [11, 12], such as Src family kinases , as well as regulating receptor recycling and degradation . Like other GPI-APs, Ly6 proteins are thus physically located in an ideal position from which to regulate or coactivate cell signaling via receptor-ligand binding or other protein-protein interactions; however, the exact molecular mechanism by which these proteins act remains unclear.
Sca-1 is the most common marker used to enrich adult murine hematopoietic stem cells (HSCs) [14, 15] and can be used to isolate a nearly pure HSC population when used in conjunction with additional markers [16, 17]. Not surprisingly, embryonic and fetal Sca-1 expression is consistent with the emergence and expansion of definitive HSCs, being first observed at E9 in the ventral dorsal aorta, with continued expression in the endothelial layer of the E11 ventral dorsal aorta  and in the fetal liver. Essentially all HSCs express Sca-1 in Ly6.2 strains, such as C57Bl/6 , although only 25% of HSCs express Sca-1 in Ly6.1 strains, such as BALB/c . This allele-specific expression appears to be due to post-transcriptional regulation or variable protein expression levels, as is suggested by studies in Sca-1 transgenic reporter mice [21, –23].
Sca-1 expression is regulated in a complex fashion in hematopoietic ontology. As HSCs differentiate into common myeloid progenitors, Sca-1 expression is downregulated , becoming upregulated on a proportion of spleen colony-forming unit (CFU-S) and culture colony-forming unit (CFU-C) progenitors . As HSCs commit to lymphoid progenitors, Sca-1 expression decreases  and prothymocytes that seed the thymic cortex upregulate expression of both Sca-1 and Sca-2, another Ly6 gene . Immature thymocytes turn off Sca-1 expression but then re-express it on mature single-positive medullary thymocytes and peripheral T cells . Sca-1 expression is further upregulated in activated lymphocytes  and in the presence of interferon-α/β and interferon-γ, whereas tumor necrosis factor-α or anti-Fas antibody downregulate Sca-1 .
Outside the hematopoietic system, Sca-1 is similarly expressed by a mixture of stem, progenitor, and differentiated cell types in a wide variety of tissues and organs. Thus, Sca-1 is used routinely in combination with negative selection against mature markers for enrichment of stem and progenitor cells. With the exception of flow cytometry (fluorescence-activated cell sorting), traditional methods for detecting this Sca-1 expression have posed many challenges. Redundancy among Ly6 genes prevents Northern analysis from being useful and requires exceptionally stringent reverse transcription- or quantitative-polymerase chain reaction conditions, whereas various Sca-1 antibodies are less than ideal for Western analysis and immunostaining. Transgenic mouse lines using a Sca-1 promoter driving LacZ [21, 23] or green fluorescent protein  expression as well as an enhanced green fluorescent protein (eGFP) knock in  have thus proved instrumental in facilitating more detailed expression studies. Overall, Sca-1 is expressed in the hematopoietic system, bone marrow, kidney, uterus, brain, mammary gland, prostate, liver, lung, pancreas, skin, thymus, lymph nodes, spleen, muscle, and bone of adult mice as well as the aorta-gonad-mesonephros, yolk sack, limb buds, urogenital ridge, liver, and hindgut region of midgestation embryos.
Sca-1 Expression Associated with Tissue-Resident Stem and Progenitor Cells
Using strategies based upon HSC enrichment, many groups are attempting to prospectively isolate and characterize stem/progenitor cells from a variety of tissues. To date, Sca-1 expression has been identified on putative stem/progenitor cell populations within the skeletal system [31, 32], mammary gland , prostate [34, 35], dermis [36, –38], skeletal muscle [39, 40], heart [41, –43], and liver [44, 45]. However, whether these Sca-1 positive populations are truly tissue-specific precursor/stem cells or represent hematopoietic, mesenchymal, or endothelial precursor/stem cells associated with these tissues is not known in all cases. Intriguingly, Sca-1 is also upregulated in a variety of murine tumors, consistent with the cancer stem cell theory.
Often, Sca-1 is coexpressed with side population (SP) activity, another phenotypic marker used to enrich stem/progenitor cells. SP cells efflux Hoechst dye via the ATP-binding cassette transporter protein ABCG2, thus appearing on the side of the FACS bulk population. Like Sca-1 expression, SP activity enriches for HSCs . Although unlikely to be universal stem/progenitor cell markers, Sca-1 expression and/or SP activity are often used as candidate markers to fractionate cell populations to test for stem/progenitor cell characteristics. However, marker expression without accompanying functional assays, such as clonal multipotentiality and self-renewal, is largely meaningless. Additionally, in situ localization analysis of marker expression within the tissue is crucial to distinguish between tissue-specific resident stem/progenitor cells and multipotent hematopoietic, endothelial, or mesenchymal progenitors associated with the tissue vasculature and circulation, all of which express Sca-1.
Although Sca-1 is widely used to prospectively isolate stem cells from various tissues, in many systems robust in vitro and in vivo functional stem cell assays have yet to be developed, making it difficult to discern between bona fide stem cells and their committed progeny (i.e., those with a lower capacity for self-renewal and higher probability of undergoing terminal differentiation). Furthermore, controversies remain as to the nature, function, and/or origin of the prospective stem/progenitor populations isolated from certain tissues using presently available methods. The following is thus intended to summarize the available evidence, given current state of the art techniques, linking Sca-1 expression with stem/progenitor cell activity (Table 1) and cancer.
Adherent marrow-derived mesenchymal stem cells/mesenchymal progenitor cells (MSC/MPC), although largely heterogeneous, express Sca-1 together with the adhesion molecules CD29 and CD44 and show no expression of hematopoietic and endothelial markers such as CD45, CD11b, and CD31. Subpopulations of these progenitors have been demonstrated to differentiate both in vitro and in vivo into lineages including bone, fat, cartilage, and muscle and have contributed to in vivo repair of bone and cartilage defects [47, , –50]. More recently, compact bone itself has been touted as a richer source of mesenchymal progenitors, with bone-derived Sca-1+CD45−CD31− cells exhibiting trilineage osteoblastic, adipocytic, and chondrocytic differentiation ability and higher clonogenic efficiencies [32, 51].
Although it remains controversial whether skeletal muscle satellite (stem) cells express Sca-1 [52, , –55], other populations of progenitors have been isolated from skeletal muscle [53, 56, –58], a high proportion of which express Sca-1 . For example, Sca-1+CD45−c-Kit− cells are believed to be nonhematopoietic in origin and can differentiate into skeletal muscle both in vitro and in vivo when injected into dystrophic mdx mice and can also give rise to hematopoietic cells as well as mesenchymal cells, such as osteoblasts [39, 40, 59]. Whether this population of Sca-1-expressing nonhematopoietic cells is distinct from bone marrow-derived MSC remains to be elucidated.
FACS analysis of dissociated murine prostate tissue revealed Sca-1 expression in 15% of cells , with higher Sca-1 expression in the proximal region . Transplantation of these Sca-1+ prostate cells into nonobese diabetic/severe combined immunodeficient mice led to significantly greater in vivo proliferative capacity, forming far more single-cell-derived multilineage tubule structures than in the Sca-1− fraction [34, 35]. Intriguingly, a sixfold increase in Sca-1+ prostatic cells was observed after castration . Thus, Sca-1 can be used to enrich for murine prostate cells that display many stem/progenitor cell-like properties.
The neonatal and adult dermis contains neural crest-derived multipotent cells called skin-derived precursors (SKPs). SKPs express Sca-1, nestin, and fibronectin and can differentiate in vitro into mesodermal cells (smooth muscle and adipocytes) as well as in vitro and in vivo into neural cells (glia, Schwann cells, and neurons) [36, 38, 60]. In vitro, SKP spheres demonstrate clonal multipotentiality and self-renewal capacity [36, 38]. Significantly, when primary mouse skin cells are sorted for Sca-1 expression, all sphere-forming ability is found within the Sca-1+ population .
Recent research has suggested that latent populations of cardiac precursors exist in the adult myocardium. More than 93% of adult cardiac SP cells are Sca-1+CD45−CD34−, exhibit levels of telomerase expression similar to neonatal myocardium, and express cardiogenic transcription factors and do not express cardiac structural genes until stimulated to differentiate in vitro . Importantly, when transplanted following ischemia/reperfusion injury, Sca-1+ cells homed to and persisted in the infarct border zone up to 2 weeks, showing 200-fold greater numbers than Sca-1− controls. Two other groups have similarly isolated Sca-1+ cells from the adult and neonatal myocardium, which not only differentiated to express cardiomyocyte proteins in vitro, but also exhibited adipogenic and osteogenic potential [42, 43]. However, in all cases the selected Sca-1+ populations differed in their gene expression profiles.
Sca-1+ cells may play a regenerative role following myocardial infarction (MI). The number of endogenous Sca-1+CD31− cells increased significantly 7 days after MI, as did the expression of Sca-1 protein . Transplantation of Sca-1+CD31− cardiac cells into the peri-infarct zone attenuated post-MI left ventricular structural and functional remodelling compared with hearts transplanted with Sca-1−CD31− cells and sham operated controls . Although a fraction of the cells appeared to differentiate into cardiomyocytes, it is unclear if the functional improvement was due to newly differentiated cardiomyocytes or due to paracrine-mediated angiogenesis, as has been demonstrated in other cardiac regeneration models . Intriguingly, at least two additional and distinct populations of progenitor-like cells, expressing c-Kit or Islet1 rather than Sca-1, have been isolated from the heart [63, 64]. It thus remains to be determined how or whether these populations are related, and which marker(s) will be best for isolation of therapeutically relevant cardiac progenitors.
Although the origin and function of endothelial progenitor cells remain controversial, they represent another population of cardiovascular progenitors with possible therapeutic applications. Bone marrow cells expressing Sca-1 and Flk1, for example, have demonstrated endothelial differentiation both in vitro and in vivo [65, 66]. Although little to no Sca-1 expression is found in the endothelium of large arteries under homeostatic conditions, upon nondenuding endothelial injury via lipopolysaccharide, the number of Sca-1+BS1 lectin+CD45− cells in the endothelial layer of the thoracic aorta dramatically increases . Interestingly, apoE-deficient mice which model arteriosclerosis have elevated numbers of Sca-1+ cells in the thoracic aorta endothelium .
Rather than the hepatocyte proliferation that accompanies other forms of liver damage, hepatic toxins induce regeneration by bipotent hepatic oval cells, which differentiate into hepatocytes or bile duct epithelial cells [68, 69]. Oval cells are clonogenic Sca-1+ cells , presumed to be transit-amplifying cells, found in the canals of Hering. Sca-1+ SP cells have also been isolated from the murine liver, of which a subset have been reported to form mixed hematopoietic and hepatocytic colonies in vitro and contribute to hepatocyte and biliary population when injected into the liver in vivo . Whether these SP cells are distinct from HSCs remains a matter of debate.
FACS analysis revealed that 20% of murine mammary epithelial cells express Sca-1, as do 75% of SP cells . Sca-1+ cells did not express the differentiation markers progesterone receptor or peanut lectin, and likely reflect a less mature mammary epithelial progenitor population . Most importantly, transplantation of Sca-1+ cells at limiting dilution resulted in increased mammary outgrowth formation rates . However, more recently, neither SP nor Sca-1high cells were found to be enriched in the population of Lin−CD29highCD24+ mammary cells, which demonstrated single cell-derived in vivo reconstitution of an entire mammary gland . In fact, Sca-1lo cells generated more mammary repopulating units than their Sca-1high counterparts . The authors posited that the discrepancy observed between their observations and those of Welm and colleagues was due to transplantation of freshly isolated versus cultured cells, with mammary epithelial Sca-1 expression increasing after several days in vitro .
In many tissues where Sca-1 is expressed, it remains unclear whether Sca-1+ cells include tissue resident progenitors. Sca-1 expression and the SP phenotype overlap substantially in the lung (although it is unclear whether these SP Sca-1+ cells express CD45 [71, 72]), yet no Sca-1 staining has been observed in the airway epithelium or in type II alveolar epithelial cells, which are believed to have progenitor activity. Recently, however, Sca-1+CD34+CD45−CD31− bronchioalveolar cells exhibiting in vitro multilineage differentiation ability have been isolated from murine lungs . Sca-1 is also expressed within neurospheres derived in the presence of lysophosphatidic acid . Nonetheless, since embryonic telencephalon-derived CNS neurospheres grown in more conventional epidermal growth factor and fibroblast growth factor-2 media do not express Sca-1 , it is unlikely that Sca-1 is expressed on brain-derived neural progenitors.
It is crucial to note that there are many tissues where Sca-1 expression and/or SP activity have been demonstrated not to be associated with progenitor activity. In the testis, for example, conflicting transplantation studies [75, –77] led to debate regarding whether SP cells, a great proportion of which also express Sca-1, are spermatogonial stem cells. Recent immunohistochemical analysis, however, revealed that Sca-1 expression was not found in the seminiferous tubules where spermatogonial stem cells reside, but was limited to the interstitial tissue . Similarly, although much of the interfollicular epidermis appears Sca-1+ positive via immunostaining, the bulge region below the sebaceous gland in the hair follicle (i.e., where epidermal stem cells reside) is Sca-1− [79, 80]. Finally, although FACS sorting of pancreatic cells revealed that ∼9% of islet and ∼15% of ductal cells express Sca-1, Sca-1+ cells did not form pancreatic colonies in vitro .
Sca-1 Expression in Cancer
A variety of murine cancers including retinoblastoma  and tumors of the mammary gland  and prostate  exhibit upregulation of Sca-1 or other Ly6 proteins. Sca-1 expression is often observed in mammary tumors induced by Wnt1 mutations; for example, 50% of hyperplastic mammary gland and mammary tumor cells from mouse mammary tumor virus (MMTV):Wnt1 transgenic mice express Sca-1. By contrast, mammary tumor cells from transgenic mice with non-Wnt mutations, such as those in MMTV:Neu and MMTV:PyMT, are less than 10% Sca-1+ . In the prostate, AKT overexpression in Sca-1+ cells initiates tumorigenesis, with cancer progression correlating with increased Sca-1+ cells . In humans, the Ly6 protein prostate stem cell antigen (PSCA) is detected in 94% and overexpressed in approximately 40% of clinically localized prostate cancers, with elevated PSCA levels correlating with increased tumor stage . In several murine tumors, higher levels of Ly6 family protein expression have been correlated with a higher malignancy phenotype (reviewed in ). Fibroblastic tumor and mammary adenocarcinoma cells expressing high Sca-1 levels are significantly more malignant than those expressing lower levels [29, 86, 87]. Similarly, DA3 tumor cells, which expressed high levels of Sca-1, were also found to express higher levels of the Ly6 protein urokinase plasminogen activator, also linked to tumor formation and metastasis .
It is widely believed that receptor-ligand interactions underlie the function of many Ly6 proteins, potentially mediating cell-cell adhesion and signaling. Overexpression of Sca-1 (CD2:Ly6a.2) in mouse T cells led to self-aggregation and adherence to nontransgenic B and T cells in vitro . These cell-cell interactions were inhibited by phosphatidylinositol-specific phospholipase C treatment (which cleaves GPI-AP from the cell surface) and by anti-Sca-1 antibodies, implying lymphocytes express a Sca-1 ligand. Similarly, Sca-1/IgM heavy chain fusion proteins were found to bind to B cells and correlated with CD22 expression . However, the observed cell-cell interactions in both studies were of low affinity, and neither study was able to detect binding between cells expressing endogenous levels of Sca-1. The discovery of a hybridoma-derived antibody that both blocked transgenic Ly6a.2 thymocyte aggregation and coimmunoprecipitated Sca-1 along with an unidentified 66 kDa protein tantalizingly suggested that a prospective Sca-1 ligand had been identified . Yet, more than six years later, the identity of this 66 kDa protein is still a mystery. Thus, the evidence is not convincing that Sca-1 acts via traditional receptor-ligand binding. Alternatively, Sca-1 could potentially coregulate a variety of different signaling pathways by modulating lipid raft composition via weak protein-protein interactions that sequester key signaling molecules. With no Sca-1-specific ligand identified, many studies utilized anti-Sca-1 antibody cross-linking to analyze Sca-1 function, often yielding conflicting results that likely do not reflect the physiological role of Sca-1. In certain contexts, for example, cross-linking Sca-1 using monoclonal antibodies led to T cell activation via an interleukin (IL)-2 autocrine signaling pathway [92, 93], whereas in other experiments antibody cross-linking in T cells downregulated IL-2 production [94, 95].
The generation of Sca-1 null mice by our lab  and the eGFP knock in mice generated by Timothy Graubert's lab  have enabled a more physiologically relevant analysis of Sca-1 function in a variety of tissues. Although both alleles of Sca-1 null mice appear phenotypically normal, careful phenotyping has demonstrated a number of defects associated with tissue-resident stem and progenitor cell populations. It is important to note that subtle differences between the two alleles of Sca-1 deficient mice may exist due to the different strategies employed in their creation, and that these dissimilarities may be magnified on various background strains.
Contrary to most expectations, based on cross-linking experiments, that Sca-1 induces lymphocyte proliferation, analysis of Sca-1 null mice demonstrated that Sca-1 functions to downregulate T-cell proliferation. Sca-1 null T cells exhibited significantly higher and more prolonged proliferation in response to T cell receptor-mediated activation, both in vitro and in vivo . Similarly, Sca-1−/− splenocytes generated more cytotoxic lymphocytes when cocultured with alloantigens . These data support a cell-signaling role for Sca-1. Sca-1 is also required for the immunosuppressive function of CD4–8− T cells and the induction of major histocompatibility complex-mismatched transplantation tolerance .
Hematopoiesis has been analyzed in both strains of Sca-1 null mice. Sca-1−/− mice demonstrated some minor hematopoietic lineage skewing, including reduced platelets and megakaryocytes , which was enhanced by bone marrow transplantation analysis that also revealed a B-cell deficiency [98, 99]. Gain of function analysis performed by Bradfute and colleagues also demonstrated a role for Sca-1 in lineage specification; Sca-1 overexpression in mouse whole bone marrow cells and human Lin−CD34+ hematopoietic precursors abrogated myeloid colony formation in both species . Although the mechanism by which Sca-1 influences cell fate determination remains unknown, we hypothesize that this occurs through altered cell signaling in lipid rafts, as discussed below in Enigmas.
In vitro (CFU-granulocyte-erythroid-macrophage-megakaryocyte) and in vivo (CFU-S) clonogenic assays revealed a slight reduction in the frequency of immature Sca-1−/− hematopoietic progenitors . Analysis of HSCs by competitive repopulation assays in both mouse strains demonstrated that Sca-1−/− cells exhibit a competitive disadvantage [98, 99]. Examination of peripheral blood chimerism in competitively transplanted animals also revealed impaired short-term Sca-1−/− repopulating ability, yet this deficiency did not persist past 4 months [98, 99]. Secondary Sca-1−/− marrow transplants by our lab, meanwhile, failed to rescue one-third of lethally irradiated wild-type hosts and resulted in only 45% engraftment in surviving animals , strongly suggesting a significant HSC self-renewal defect in Sca-1−/− mice. However, Bradfute et al. suggested that the observed engraftment reduction may, at least in part, be due to a Sca-1−/− homing defect. Indeed, 24 hours post-transplantation, Sca-1−/− marrow cells formed significantly fewer in vitro CFU-C colonies than control cells . To evaluate HSC self-renewal in the absence of homing effects, Bradfute and colleagues challenged bone marrow chimeras to three rounds of 5-fluorouracil (FU) injections, killing cycling cells, over a 10-week period. Since the percentage of Sca-1−/− peripheral blood cells remained unchanged, the authors concluded that Sca-1 was unnecessary for HSC self-renewal in this context. However, this protocol has not been validated as a test for self-renewal capacity; thus, our lab is currently performing mosaic analysis by embryo↔embryo aggregations to re-evaluate Sca-1−/− HSC self-renewal in a competition assay independent of cell homing effects.
More striking than the defects in hematopoiesis, Sca-1−/− mice exhibit age-related osteoporosis characterized by deterioration in bone material, microarchitectural, and mechanical properties ( and C. Holmes, manuscript submitted for publication). As is the case with human senile (type II) osteoporosis, these skeletal deficiencies are due to decreased bone formation rather than increased bone resorption. In fact, Sca-1−/− mice display reduced in vitro osteoclastogenesis due to both cell-intrinsic defects (C. Holmes, manuscript submitted for publication) and a reduced ability of mutant bone marrow stromal cells to support osteoclast differentiation . Early in life, Sca-1−/− bone marrow contains a higher frequency of MPCs than wild-type controls, with a concomitant increase in osteoprogenitors; however, by 7 months of age, a dramatic decrease in Sca-1−/− MPCs is exhibited, resulting in decreased osteoprogenitors, osteoblasts, and bone formation, thus leading to reduced bone quality and strength (C. Holmes, T.S. Khan, C. Owen, N. Ciliberti, M.D. Grynpas, W.L. Stanford, manuscript submitted for publication). Because robust protocols for MPC transplantation have yet to be developed, we evaluated self-renewal capacity in vitro and found a profound defect in the ability of Sca-1−/− MPCs to be serially passaged . Interestingly, decreased unmineralized bone matrix is observed in Sca-1−/− bones at 5 months of age despite increased osteoprogenitors, implying defective osteoblast matrix production. This reduced bone formation may create a feedback loop that causes the observed increase in Sca-1−/− MPCs early in life, which, in turn, exacerbates the self-renewal defect by inducing stem cell cycling, thus leading to depletion of the Sca-1 null MPC pool.
Evidence from Sca-1 null mice and studies utilizing the C2C12 skeletal myoblast cell line have together revealed that Sca-1 plays a role in myoblast differentiation, proliferation, fusion, and cell-cycle exit. In C2C12 cells, expression of Sca-1 is transiently upregulated coincident with cell cycle withdrawal and the initiation of differentiation [101, 102]. Blocking Sca-1 by antibodies or downregulating expression via antisense inhibited myoblast fusion and promoted proliferation . Likewise, primary Sca-1+ myoblasts divided slower and formed myotubes less readily than their Sca-1− counterparts, whereas primary myoblast cultures from 3–5-month-old eGFP knock in Sca-1−/− mice exhibited increased proliferation and reduced numbers of undifferentiated cells . Further analysis revealed that Sca-1 regulates myofiber size in an age-dependent manner that closely mirrors the skeletal phenotype, with increased muscle size observed in young (2–4-month-old) Sca-1 null mice and reduced muscle size exhibited by older (12-month-old) Sca-1−/− mice compared with controls . Although reduced myofiber size in older Sca-1 null mice was not accompanied by changes in the numbers of CD34+ satellite cells, the authors did not evaluate whether larger muscle size is accompanied by an early increase in muscle progenitors . Further analysis of other, particularly earlier, time points would thus be of great interest. An abnormal proliferative response to physiological muscle loading conditions cannot be ruled out and should also be explored. Similarly, we have observed that Sca-1−/− mice may respond abnormally to muscle injury (P. Bonyadi and W.L. Stanford, unpublished data). Thus, overall Sca-1 appears to downregulate muscle cell proliferation, thereby maintaining a pool of functional progenitor cells for muscle homeostasis and repair.
Thorough analysis of other organ systems is pending; however, to date, the majority of Sca-1 null phenotypes appears to be associated with contexts that stress tissue-resident stem/progenitor cells, such as aging, transplantation, and injury. In the case of the muscle phenotype, for example, enhanced myoblast proliferation may be the underlying cause of the larger muscle fibers observed in young animals; however, this proliferation also likely diminishes the pool of available myogenic progenitors, resulting in the diminished muscle size of older animals. Similarly, mesenchymal- and osteoprogenitor populations peak early and are unable to maintain a pool of osteoprogenitors later in life. Impaired Sca-1−/− HSC transplantation engraftment and muscle regeneration ability, furthermore, imply diminished stress responses. Stress forces more stem cells to undergo division and, thus, make fate “decisions.” We hypothesize that, in the absence of Sca-1, differentiation signals are favored at the expense of self-renewal, which over time may lead to stem/progenitor exhaustion.
Aging has been associated with qualitative and quantitative changes in stem cells, with HSC aging linked to a slower and incomplete response to hematopoietic stress [103, –105]. Sca-1 deficiency appears to hasten the onset of these changes. Oxidative stress, meanwhile, leads to premature HSC “aging” via increased p38 mitogen-activated protein kinase signaling, pushing HSCs from quiescence to proliferation, leading to progenitor exhaustion over time . Nitric oxide synthase-mediated suppression of hematopoietic marrow colony formation has been demonstrated via Sca-1 monoclonal antibody cross-linking studies, which, although not reflective of physiological function, suggests a link between Sca-1 and oxidative stress responses .
How Does Sca-1 Mediate Stem Cell Signaling?
As a GPI-AP lacking an identified ligand, Sca-1 likely acts as a coregulator of lipid raft signaling and, in turn, stem cell fate decisions. We postulate that Sca-1 alters lipid raft composition via weak protein-protein interactions, sequestering or obstructing key signaling molecules in the vicinity of their receptors or even promoting raft clustering. According to the ligand/receptor signaling threshold stem cell model , a threshold level of signaling-competent cytokine-receptor complexes is required for self-renewal. We hypothesize that Sca-1 deficiency reduces the numbers of functional signaling complexes or alters signaling dynamics in such a manner that fewer stem cells reach this signaling threshold, thus leading to increased differentiation and exhaustion of the stem cell pool with time (Fig. 1). Altered lipid raft signaling could change the number of signaling-competent cytokine-receptor complexes by a number of mechanisms. For example, caveolin-mediated receptor internalization associated with lipid raft signaling leads to ubiquitination and downregulation of the signal, whereas clathrin-mediated receptor internalization promotes receptor recycling and signal propagation . Thus, if Sca-1 could affect the ratio of signaling complexes found within lipid rafts versus nonraft membrane domains, then Sca-1 would affect the propagation of a signal. Furthermore, we propose that tissue-resident stem cells use tissue-specific primary signaling molecules such as tyrosine kinases, whereas common cosignaling molecules, such as Sca-1, are conserved across many tissues and function to modify the balance between self-renewal and differentiation, especially under conditions of stress such as tissue regeneration. Consistent with our model, we have observed differential phosphotyrosine signatures in lipid rafts of Sca-1 null versus wild-type mast cells in response to various signals (N. Ciliberti and W.L. Stanford, unpublished observations).
Through Which Downstream Pathways Does Sca-1 Act?
Sca-1 could potentially play a role in several different lipid raft-mediated signaling pathways, including those involving receptor tyrosine kinases and Src family kinases. Exhibiting a close biochemical association , mutations in GPI-APs and Src family kinases often lead to opposing phenotypes in mice. For example, in contrast to the osteoporosis in Sca-1−/− mice, Src−/− animals display osteopetrosis due to both impaired osteoclast function  and increased osteoblast differentiation . Similarly, Sca-1 and Src family kinase mutants display opposing lymphoid phenotypes [112, –114]. More importantly, in vitro analyses suggest that the effects of Sca-1 on T-cell activation and myoblast differentiation are mediated, at least in part, via Fyn signaling. Downregulation of Sca-1 via antisense expression in T-cell lines, for example, leads to impaired T-cell receptor (TCR)-β chain transcription and impaired Fyn activity . Meanwhile, Sca-1 antisense expression in C2C12 myoblasts leads to premature activation of Fyn accompanied by sustained proliferation and reduced differentiation, a phenotype that is rescued when these cells are transfected with a dominant negative form of Fyn . Correspondingly, transfection of wild-type cells with active Fyn produced the Sca-1 antisense phenotype . Overall, current evidence suggests that Sca-1 may serve as a negative regulator of Src family kinase signaling.
Sca-1 and lipid rafts also demonstrate close associations with c-Kit signaling in hematopoietic stem and progenitor cells. For instance, after ligand binding, c-Kit translocates to clustered lipid rafts . More importantly, compound Sca-1−/−;c-Kit mutant (Wv/Wv) mice demonstrated dramatically reduced viability, demonstrating genetic interaction and suggesting biochemical interaction between Sca-1 and c-Kit . Finally, after 5-FU injection, Sca-1−/− cells exhibited a decreased percentage of c-Kit+Lin− cells .
Does Sca-1 Have a Human Ortholog?
A number of potential human homologs for murine Ly6 family proteins have been identified (reviewed in ), many of which are encoded by genes localized to a region of chromosome 8, specifically 8q24.3 , which is syntenic to the region where the mouse Ly6 locus resides. Sca-1, however, does not have a human homolog; in fact, a 500-kilobase region of the Ly6 locus of mouse chromosome 15 encoding nine genes including Sca-1 and five additional Ly6 genes was deleted between mouse and rat speciation (Fig. 2). Interestingly, the Ly6 genes flanking this deleted region are conserved between rat and human. Considering the wide range of Sca-1 tissue expression and the important roles it plays in mediating stem cell stress responses in mice, this evolutionary loss of Sca-1 appears puzzling. Although it is possible that the precise function(s) of Sca-1 have been rendered less important during evolution, it is likely that at least some of the roles played by Sca-1 in mice are assumed by one or a number of human Ly6 proteins or other GPI-APs.
Consistent with this speculation, the human Ly6 family proteins characterized to date exhibit a diverse array of expression and functions, many of which are linked to human cancers, reminiscent of the association between Sca-1 and murine cancers. Furthermore, there are functional similarities between Sca-1 and other GPI-APs. For example, Thy-1−/− mice exhibit enhanced TCR signaling  and impaired cutaneous immune response  reminiscent of the T-cell phenotypes of Sca-1−/− mice, and PrP null mice exhibit reduced HSC self-renewal . Regardless of whether the human Sca-1 ortholog proves to be a Ly6 protein or other GPI-AP, the theory that disruption of the signaling dynamics that mediate stem cell self-renewal and/or quiescence can lead to progenitor exhaustion is unquestionably relevant to human degenerative diseases.
Although a human ortholog of Sca-1 has yet to be identified, analysis of Sca-1 mutant mice to date has demonstrated a number of important stem cell concepts that are equally relevant to human health and disease, including the importance of mesenchymal stem and progenitor cells to bone homeostasis and deterioration, the association between degenerative diseases and exhaustion of the stem cell pool, and the importance of the stem cell stress responses to tissue maintenance. Thus, the in-depth study of the mechanisms that underlie Sca-1 function in mice is still crucial to further our understanding of Ly6 family proteins, GPI-APs, and, most importantly, human stem cell biology. Once an ortholog and/or the pathways responsible for Sca-1 function(s) in humans have been identified, we will have a highly attractive drug target for the manipulation of tissue-resident stem cell pools enabling numerous new therapeutic and regenerative medicine applications.
Disclosure of Potential Conflicts of Interest
The authors indicate no potential conflicts of interest.
We thank C.E. Kandel for his help with Figure 2 and T.S. Khan for his insightful comments. W.L.S. is a Canadian Research Chair in Stem Cell Biology & Functional Genomics. Our Sca-1 research has been supported by the Canadian Institutes of Health Research and its Institute of Musculoskeletal Health and Arthritis and Institute of Infection and Immunity. We apologize to the many colleagues who have provided valuable insights and large amounts of data within the Ly6/Sca-1 field whose manuscripts were not cited because of strict space limitations.