Glutamate transporter Slc1a3 mediates inter‐niche stem cell activation during skin growth

Tissues contain distinct stem cell niches, but whether cell turnover is coordinated between niches during growth is unknown. Here, we report that in mouse skin, hair growth is accompanied by sebaceous gland and interfollicular epidermis expansion. During hair growth, cells in the bulge and outer root sheath temporarily upregulate the glutamate transporter SLC1A3, and the number of SLC1A3⁺ basal cells in interfollicular epidermis and sebaceous gland increases. Fate mapping of SLC1A3⁺ cells in mice revealed transient expression in proliferating stem/progenitor cells in all three niches. Deletion of slc1a3 delays hair follicle anagen entry, uncouples interfollicular epidermis and sebaceous gland expansion from the hair cycle, and leads to reduced fur density in aged mice, indicating a role of SLC1A3 in stem/progenitor cell activation. Modulation of metabotropic glutamate receptor 5 activity mimics the effects of SLC1A3 deletion or inhibition. These data reveal that stem/progenitor cell activation is synchronized over distinct niches during growth and identify SLC1A3 as a general marker and effector of activated epithelial stem/progenitor cells throughout the skin.     

Basal Keratinocytes Contribute to All Strata of the Adult Zebrafish Epidermis

The epidermis of terrestrial vertebrates is a stratified epithelium and forms an essential protective barrier. It is continually renewed, with dead corneocytes shed from the surface and replaced from a basal keratinocyte stem cell population. Whilst mouse is the prime model system used for epidermal studies, there is increasing employment of the zebrafish to analyse epidermis development and homeostasis, however the architecture and ontogeny of the epidermis in this system are incompletely described. In particular, it is unclear if adult zebrafish epidermis is derived entirely from the basal epidermal stem cell layer, as in the mouse, or if the most superficial keratinocyte layer is a remnant of the embryonic periderm. Furthermore, a relative paucity of cellular markers and genetic reagents to label and manipulate the basal epidermal stem cell compartment has hampered research. Here we show that the type I keratin, krtt1c19e, is a suitable marker of the basal epidermal layer and identify a krtt1c19e promoter fragment able to drive strong and specific expression in this cell type. Use of this promoter to express an inducible Cre recombinase allowed permanent labelling of basal cells during embryogenesis, and demonstrated that these cells do indeed generate keratinocytes of all strata in the adult epidermis. Further deployment of the Cre-Lox system highlighted the transient nature of the embryonic periderm. We thus show that the epidermis of adult zebrafish, as in the mouse, derives from basal stem cells, further expanding the similarities of epidermal ontogeny across vertebrates. Future use of this promoter will assist genetic analysis of basal keratinocyte biology in zebrafish.     

Epidermal stem cells: interactions in developmental environments

Homeostasis of continuously renewing adult tissues, such as the epidermis of the skin, is maintained by epidermal stem cells (EpiSC), which are a small population of undifferentiated, self-renewing basal keratinocyte cells that produce daughter transit amplifying (TA) cells to make up the majority of the proliferative basal cell population in the epidermis. We have isolated EpiSC from neonatal and adult skin, and shown that these cells can regenerate an epidermis that lasts long term in vitro and in vivo, and that permanently expresses a recombinant gene in the regenerated tissue (Bickenbach and Dunnwald, 2000; Dunnwald et al., 2001). When we injected murine EpiSC into the developing blastocyst environment of the mouse, we found that both neonatal and adult EpiSC retained some ability to participate in the formation of tissues from all three germ layers (Liang and Bickenbach, 2002; Bickenbach and Chinnathambi, 2004; Liang et al., 2004). Although it appears evident that EpiSC act as pluripotent stem cells, how this reprogramming takes place is not understood. EpiSC might directly transdifferentiate into other cell types or they might first dedifferentiate into a more primitive cell type, and then proceed to develop along a cell lineage pathway. To begin to unravel this, we co-cultured EpiSC with embryonic stem (ES) cells, and found that EpiSC could alter their cell lineage protein expression to that of a more primitive cell type. We also placed EpiSC in a wounded environment and found that EpiSC interacted with the mesenchymal cells repopulating the wound bed. Our findings indicate that the population of cells that we isolate as EpiSC has a pluripotent capability. This has led us to postulate a paradigm shift for somatic stem cells. We propose that tissues maintain a sequestered population of uncommitted stem cells that retain a regenerative response which is enhanced when the cells are exposed to developmental or stress influences.     

The spatial relationship between stem cells and their progeny in the basal layer of human epidermis: a new view based on whole-mount labelling and lineage analysis

In order to examine the spatial organisation of stem cells and their progeny in human epidermis, we developed a method for whole-mount epidermal immunofluorescence labelling using high surface beta1 integrin expression as a stem cell marker. We confirmed that there are clusters of high beta1 integrin-expressing cells at the tips of the dermal papillae in epidermis from several body sites, whereas alpha6 integrin expression is more uniform. The majority of actively cycling cells detected by Ki67 or bromodeoxyuridine labelling were found in the beta1 integrin-dull, transit amplifying population and integrin-negative, keratin 10-positive cells left the basal layer exclusively from this compartment. When we examined p53-positive clones in sun-exposed epidermis, we found two types of clone that differed in size and position in a way that was consistent with the founder cell being a stem or transit amplifying cell. The patterning of the basal layer implies that transit amplifying cells migrate over the basement membrane away from the stem cell clusters. In support of this, isolated beta1 integrin-dull keratinocytes were more motile on type IV collagen than beta1 integrin-bright keratinocytes and EGFP-labelled stem cell clones in confluent cultured sheets were compact, whereas transit amplifying clones were dispersed. The combination of whole-mount labelling and lineage marking thus reveals features of epidermal organisation that were previously unrecognised.     

Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing

Several recent studies suggest the isolation of stem cells in skeletal muscle, but the functional properties of these muscle-derived stem cells is still unclear. In the present study, we report the purification of muscle-derived stem cells from the mdx mouse, an animal model for Duchenne muscular dystrophy. We show that enrichment of desmin(+) cells using the preplate technique from mouse primary muscle cell culture also enriches a cell population expressing CD34 and Bcl-2. The CD34(+) cells and Bcl-2(+) cells were found to reside within the basal lamina, where satellite cells are normally found. Clonal isolation and characterization from this CD34(+)Bcl-2(+) enriched population yielded a putative muscle-derived stem cell, mc13, that is capable of differentiating into both myogenic and osteogenic lineage in vitro and in vivo. The mc13 cells are c-kit and CD45 negative and express: desmin, c-met and MNF, three markers expressed in early myogenic progenitors; Flk-1, a mouse homologue of KDR recently identified in humans as a key marker in hematopoietic cells with stem cell-like characteristics; and Sca-1, a marker for both skeletal muscle and hematopoietic stem cells. Intramuscular, and more importantly, intravenous injection of mc13 cells result in muscle regeneration and partial restoration of dystrophin in mdx mice. Transplantation of mc13 cells engineered to secrete osteogenic protein differentiate in osteogenic lineage and accelerate healing of a skull defect in SCID mice. Taken together, these results suggest the isolation of a population of muscle-derived stem cells capable of improving both muscle regeneration and bone healing.     

Correlation between Musashi-1 and c-hairy-1 expression and cell proliferation activity in the developing intestine and stomach of both chicken and mouse

Musashi-1 (Msi-1) is an RNA-binding protein that plays key roles in the maintenance of neural stem cell states and in their differentiation into neural cells. Msi-1 has also been proposed as a candidate marker gene of mammalian intestinal stem cells and their immediate lineages. In this study, we examined Msi-1 expression in the small intestine and the stomach of both chicken and mouse during embryonic, fetal and postnatal development. In addition, we analyzed the expression of c-hairy-1, a chicken homologue of mouse Hes1, and assessed the proliferative activity of the cells expressing both of these factors. Significantly, during the development of these digestive organs in both species Msi-1 expression showed dynamic changes, suggesting that it is important for digestive organ development, particularly for epithelial differentiation. Based on our observations of the expression patterns of Msi-1 and c-hairy-1 in the adult small intestine, we speculate that Msi-1 is also a stem cell marker of the chicken small intestinal epithelium.     

Identification of a putative intestinal stem cell and early lineage marker; musashi-1

There are few reliable markers for adult stem cells and none for those of the intestinal epithelium. Previously, indirect experimental approaches have predicted stem cell position and numbers. The Musashi-1 (Msi-1) gene encodes an RNA binding protein associated with asymmetric divisions in neural progenitor cells. Two-day-old, adult, and 4.5 h, 1-, 2-, 4- and 12-day post-irradiation samples of BDF1 mouse small intestine, together with some samples of mouse colon were stained with a rat monoclonal antibody to Musashi-1 (14 H-1). Min ( + / - ) mice with small intestinal adenomas of varying sizes were also analysed. Samples of human small and large bowel were also studied but the antibody staining was weak. Musashi-1 expression was observed using immunohistochemistry in neonatal, adult, and regenerating crypts with a staining pattern consistent with the predicted number and distribution of early lineage cells including the functional stem cells in these situations. Early dysplastic crypts and adenomas were also strongly Musashi-1 positive. In situ hybridization studies showed similar expression patterns for the Musashi mRNA and real-time quantitative RT-PCR showed dramatically more Msi-1 mRNA expression in Min tumours compared with adjacent normal tissue. These observations suggest that Musashi-1 is a marker of stem and early lineage progenitor cells in murine intestinal tissue.     

Lineage of anuran epidermal basal cells and their differentiation potential in relation to metamorphic skin remodeling

The anuran remodels the larval epidermis into the adult one during metamorphosis. Larval and adult epidermal cells of the bullfrog were characterized by determining the presence of huge cytoplasmic keratin bundles and the expression profiles of specific marker genes, namely colalpha1 (collagen alpha1 (I)), rlk (larval keratin) and rak (adult keratin). We identified four types of epidermal basal cells: (i) basal skein cells that have keratin bundles and express colalpha1 and rlk; (ii) rak+-basal skein cells that have keratin bundles and express colalpha1, rlk, and rak; (iii) larval basal cells that express rlk and rak; and (iv) adult basal cells that express rak. These traits suggested that these basal cells are on the same lineage in which basal skein cells are the original progenitor cells that consecutively differentiate into rak+-basal skein cells into larval basal cells, and finally into adult basal cells. To directly verify the differentiation potential of larval basal cells into adult ones, the mono-layered epidermis composed of larval basal cells was cultured in the presence of aldosterone and thyroid hormone. In this culture, larval basal cells differentiated into adult basal cells that reconstituted the adult epidermis. Thus, it was concluded that larval basal cells are the direct progenitor cells of the adult epidermal stem cells.     

Sca-1 negatively regulates proliferation and differentiation of muscle cells

Satellite cells are tissue-specific stem cells critical for skeletal muscle growth and regeneration. Upon exposure to appropriate stimuli, satellite cells produce progeny myoblasts. Heterogeneity within a population of myoblasts ensures that a subset of myoblasts readily differentiate to form myotubes, whereas other myoblasts remain undifferentiated and thus available for future muscle growth. The mechanisms that contribute to this heterogeneity in myoblasts are largely unknown. We show that satellite cells are Sca-1(neg) but give rise to myoblasts that are heterogeneous for sca-1 expression. The majority of myoblasts are sca-1(neg), rapidly divide, and are capable of undergoing myogenic differentiation to form myotubes. In contrast, a minority population is sca-1(pos), divides slower, and does not readily form myotubes. Sca-1 expression is not static but rather dynamically modulated by the microenvironment. Gain-of-function and loss-of-function experiments demonstrate that sca-1 has a functional role in regulating proliferation and differentiation of myoblasts. Myofiber size of sca-1 null muscles is altered in an age-dependent manner, with increased size observed in younger mice and decreased size in older mice. These studies reveal a novel system that reversibly modulates the myogenic behavior of myoblasts. These studies provide evidence that, rather than being a fixed property, myoblast heterogeneity can be modulated by the microenvironment.     

急性呼吸窘迫综合征小鼠肺内源性干细胞表达水平的研究

目的探讨急性呼吸窘迫综合征（ARDS）小鼠肺组织中肺内源性干细胞的表达水平。 方法10只C57BL/6小鼠分成两组：实验组和对照组,实验组通过气管内注射脂多糖（LPS）构建小鼠ARDS模型,采用气管内注射PBS作为对照组;采用胶原酶、热消化法消化小鼠肺组织获取小鼠肺单细胞悬液;双重免疫荧光染色方法鉴定小鼠肺组织中sca-1⁺CD31^-CD45^-细胞;流式细胞术对肺sca-1⁺CD31^-CD45^-细胞进行分选。采用方差分析及独立t检验进行统计学分析。 结果通过气管内注入LPS成功制作小鼠急性ARDS模型;5只小鼠的全肺组织制备单细胞悬液总数目达5×10⁷个/ml,活细胞百分比为98﹪;肺内源性干细胞包括Ⅱ型肺泡上皮细胞、clara细胞以及支气管肺泡干细胞等,通过肺组织双重免疫荧光染色,验证小鼠肺组织Ⅱ型肺泡上皮细胞、clara细胞以及支气管肺泡干细胞;对照组及实验组各样本肺内源性干细胞数目占单细胞悬液细胞数比例呈正态分布,且实验组肺内源性干细胞数目水平（10.73±10.65）﹪较对照组水平（12.23±0.73）﹪降低（t = -3.405,P < 0.01）。 结论ARDS时,小鼠肺内源性干细胞（sca-1⁺CD31^-CD45^-）水平降低,减少的肺内源性干细胞具体去向尚不明确,其有可能参与机体急性炎症过程中气道上皮细胞的修复、再生过程。     

Expression profile of the stem cell markers in human Hertwig’s epithelial root sheath/Epithelial rests of Malassez cells

Hertwig’s epithelial root sheath/Epithelial rests of Malassez (HERS/ERM) cells are unique epithelial cells in the periodontal ligament. They remain in periodontal tissues through-out the adult life, and it is expected that their functional role is to maintain the homeostasis of the periodontium through reciprocal interactions with other periodontal cells. In this study, we investigated whether HERS/ERM cells have primitive stem cell characteristics: those of embryonic stem cells as well as of epithelial stem cells. Primary HERS/ERM cells had typical epithelial cell morphology and characteristics and they maintained for more than five passages. They expressed epithelial stem cell-related genes: ABCG2, ANp63, p75, EpCAM, and Bmi-1. Moreover, the expression of embryonic stem cell markers such as Oct-4, Nanog, and SSEA-4 were detected. Next, we investigated whether the expression of these stem cell markers was maintained during the sub-culture process. HERS/ERM cells showed different expression levels of these stemness genes at each passage, but their expression was maintained throughout the passages. Taken together, our data suggest that a primary culture of HERS/ERM cells contains a population of primitive stem cells that express epithelial stem cell markers and embryonic stem cell markers. Furthermore, these cell populations were maintained during the sub-culturing process in our culture conditions. Therefore, our findings suggest that there is a strong possibility of accomplishing cementum tissue engineering with HERS/ERM cells.     

Identification of stage-specific markers during differentiation of hair cells from mouse inner ear stem cells or progenitor cells in vitro

The induction of inner ear hair cells from stem cells or progenitor cells in the inner ear proceeds through a committed inner ear sensory progenitor cell stage prior to hair cell differentiation. To increase the efficacy of inducing inner ear hair cell differentiation from the stem cells or progenitor cells, it is essential to identify comprehensive markers for the stem cells/progenitor cells from the inner ear, the committed inner ear sensory progenitor cells and the differentiating hair cells to optimize induction conditions. Here, we report that we efficiently isolated and expanded the stem cells or progenitor cells from postnatal mouse cochleae, and induced the generation of inner ear progenitor cells and subsequent differentiation of hair cells. We profiled the gene expression of the stem cells or progenitor cells, the inner ear progenitor cells, and hair cells using aRNA microarray analysis. The pathway and gene ontology (GO) analysis of differentially expressed genes was performed. Analysis of genes exclusively detected in one particular cellular population revealed 30, 38, and 31 genes specific for inner ear stem cells, inner ear progenitor cells, and hair cells, respectively. We further examined the expression of these genes in vivo and determined that Gdf10+Ccdc121, Tmprss9+Orm1, and Chrna9+Espnl are marker genes specific for inner ear stem cells, inner ear progenitor cells, and differentiating hair cells, respectively. The identification of these marker genes will likely help the effort to increase the efficacy of hair cell induction from the stem cells or progenitor cells.     

Immunohistochemical analysis of DNA mismatch repair enzyme hMSH-2 in normal human skin and basal cell carcinomas

We have analysed the expression and distribution of the DNA mismatch repair enzyme hMSH-2 in normal skin and basal cell carcinomas. hMSH-2 protein was investigated immunohistochemically (normal human skin: n=10; basal cell carcinomas: n=16) on frozen sections using a highly sensitive streptavidin–peroxidase technique and a specific mouse monoclonal antibody (clone FE11). In normal human skin, we found nuclear immunoreactivity for hMSH-2 in epidermal keratinocytes of the basal and first 1–3 suprabasal cell layers. All basal cell carcinomas analysed revealed strong nuclear imunoreactivity that was pronounced in peripheral tumour cells and cells of the palisade. Expression of hMSH-2 protein was consistently and strongly upregulated in tumour cells of the carcinomas as compared to adjacent unaffected epidermis or epidermis of normal human skin. Twelve of the sixteen carcinomas analysed revealed no visual correlation in comparing the labelling patterns for hMSH-2 with the labelling pattern for the proliferation marker Ki-67. Our findings indicate that (a) hMSH-2 is expressed in human epidermal keratinocytes, predominantly in lower cell layers of the viable epidermis; (b) expression of hMSH-2 protein is strongly upregulated in basal cell carcinomas as compared to unaffected epidermis; (c) the level of hMSH-2 proteins in the carcinomas is not exclusively regulated by the proliferative activity of these tumour cells; (d) inactivating mutations of the hMSH-2 gene may in the carcinomas not be involved in the carcinogenesis or microsatellite instability secondary to replication errors; (e) expression of hMSH-2 may be of importance for the genetic stability of basal cell carcinomas in vivo.     

Keratinocyte stem cells: Friends and foes

Skin and its appendages provide a protective barrier against the assaults of the environment. To perform its role, epidermis undergoes an ongoing renewal through a balance of proliferation and differentiation/apoptosis called homeostasis. Keratinocyte stem cells reside in a special microenvironment called niche in basal epidermis, adult hair follicle, and sebaceous glands. While a definite marker has yet to be detected, data raised part in humans and part in the mouse system point to a critical role of stem and its progeny transit amplifying cells in epidermal homeostasis. Stem cells are protected from apoptosis and are long resident in adult epidermis. This renders them more prone to be the origin of skin cancer. In this review, we will outline the main features of adult stem cells in mouse and humans and discuss their fate in relation to differentiation, apoptosis, and cancer. J. Cell. Physiol. 225: 310–315, 2010. © 2010 Wiley‐Liss, Inc.     

Maintenance of distinct melanocyte populations in the interfollicular epidermis

Hair follicles and sweat glands are recognized as reservoirs of melanocyte stem cells (MSCs). Unlike differentiated melanocytes, undifferentiated MSCs do not produce melanin. They serve as a source of differentiated melanocytes for the hair follicle and contribute to the interfollicular epidermis upon wounding, exposure to ultraviolet irradiation or in remission from vitiligo, where repigmentation often spreads outwards from the hair follicles. It is unknown whether these observations reflect the normal homoeostatic mechanism of melanocyte renewal or whether unperturbed interfollicular epidermis can maintain a melanocyte population that is independent of the skin's appendages. Here, we show that mouse tail skin lacking appendages does maintain a stable melanocyte number, including a low frequency of amelanotic melanocytes, into adult life. Furthermore, we show that actively cycling differentiated melanocytes are present in postnatal skin, indicating that amelanotic melanocytes are not uniquely relied on for melanocyte homoeostasis.     

Cell differentiation lineage in the prostate

Prostatic epithelium consists mainly of luminal and basal cells, which are presumed to differentiate from common progenitor/stem cells. We hypothesize that progenitor/stem cells are highly concentrated in the embryonic urogenital sinus epithelium from which prostatic epithelial buds develop. We further hypothesize that these epithelial progenitor/stem cells are also present within the basal compartment of adult prostatic epithelium and that the spectrum of differentiation markers of embryonic and adult progenitor/stem cells will be similar. The present study demonstrates that the majority of cells in embryonic urogenital sinus epithelium and developing prostatic epithelium (rat, mouse, and human) co-expressed luminal cytokeratins 8 and 18 (CK8, CK18), the basal cell cytokeratins (CK14, CK5), p63, and the so-called transitional or intermediate cell markers, cytokeratin 19 (CK19) and glutathione-S-transferase-pi (GSTpi). The majority of luminal cells in adult rodent and human prostates only expressed luminal markers (CK8, CK18), while the basal epithelial cell compartment contained several distinct subpopulations. In the adult prostate, the predominant basal epithelial subpopulation expressed the classical basal cell markers (CK5, CK14, p63) as well as CK19 and GSTpi. However, a small fraction of adult prostatic basal epithelial cells co-expressed the full spectrum of basal and luminal epithelial cell markers (CK5, CK14, CK8, CK18, CK19, p63, GSTpi). This adult prostatic basal epithelial cell subpopulation, thus, exhibited a cell differentiation marker profile similar to that expressed in embryonic urogenital sinus epithelium. These rare adult prostatic basal epithelial cells are proposed to be the progenitor/stem cell population. Thus, we propose that at all stages (embryonic to adult) prostatic epithelial progenitor/stem cells maintain a differentiation marker profile similar to that of the original embryonic progenitor of the prostate, namely urogenital sinus epithelium. Adult progenitor/stem cells co-express both luminal cell, basal cell, and intermediate cell markers. These progenitor/stem cells differentiate into mature luminal cells by maintaining CK8 and CK18, and losing all other makers. Progenitor/stem cells also give rise to mature basal cells by maintaining CK5, CK14, p63, CK19, and GSTpi and losing K8 and K18. Thus, adult prostate basal and luminal cells are proposed to be derived from a common pleuripotent progenitor/stem cell in the basal compartment that maintains its embryonic profile of differentiation markers from embryonic to adult stages.     

角蛋白15在大鼠皮肤发育中表达的研究

目的研究角蛋白15(K15)在大鼠皮肤发育中的表达状况,定位表皮干细胞.方法以不同年龄大鼠背部皮肤为标本,用组织学方法,观察出生后大鼠皮肤的形态发育变化;以K15单克隆抗体为一抗,进行免疫组织化学染色,观察K15在大鼠皮肤中的表达状况.结果(1)组织学方法显示,随着年龄的增长,大鼠背部表皮细胞层数逐渐变少;在毛囊的生长周期中,以隆突区为界,毛囊上段为恒定区,下段呈周期性变化(2)免疫组化染色显示,毛囊隆突区细胞胞浆表达K15,随年龄的增长,K15阳性细胞出现在毛母质细胞区、毛囊外根鞘和表皮基底层.结论表皮干细胞位于毛囊隆突区,与表皮的更新和毛囊的周期性变化有关.     

Stimulation of human epidermal differentiation by delta-notch signalling at the boundaries of stem-cell clusters

BACKGROUND: Human epidermis is renewed throughout life from stem cells in the basal layer of the epidermis. Signals from the surrounding keratinocytes influence the differentiation of the stem cells, but the nature of the signals is unknown. In many developing tissues, signalling mediated by the transmembrane protein Delta1 and its receptor Notch1 inhibits differentiation. Here, we investigated the role of Delta-Notch signalling in postnatal human epidermis. RESULTS: Notch1 expression was found in all living epidermal layers, but Delta1 expression was confined to the basal layer of the epidermis, with highest expression in those regions where stem cells reside. By overexpressing Delta1 or Delta(T), a truncated form of Delta1, in primary human keratinocytes and reconstituting epidermal sheets containing mixtures of Delta-overexpressing cells and wild-type cells, we found that cells expressing high levels of Delta1 or Delta(T) failed to respond to Delta signals from their neighbours. In contrast, wild-type keratinocytes that were in contact with neighbouring cells expressing Delta1 were stimulated to leave the stem-cell compartment and initiate terminal differentiation after a few rounds of division. Delta1 promoted keratinocyte cohesiveness, whereas Delta(T) did not. CONCLUSIONS: We propose that high Delta1 expression by epidermal stem cells has three effects: a protective effect on stem cells by blocking Notch signalling; enhanced cohesiveness of stem-cell clusters, which may discourage intermingling with neighbouring cells; and signalling to cells at the edges of the clusters to differentiate. Notch signalling in epidermal stem cells thus differs from other progenitor cell populations in promoting, rather than suppressing, differentiation.     

Human and rodent bone marrow mesenchymal stem cells that express primitive stem cell markers can be directly enriched by using the CD49a molecule

Bone marrow (BM) from human and rodent species contains a population of multipotential cells referred to as mesenchymal stem cells (MSCs). Currently, MSCs are isolated indirectly by using a culture step and then the generation of fibroblast colony-forming units (CFU-fs). Unprocessed or native BM MSCs have not yet been fully characterised. We have previously developed a direct enrichment method for the isolation of MSCs from human BM by using the CD49a protein (alpha1-integrin subunit). As the CD49a gene is highly conserved in mammals, we have evaluated whether this direct enrichment can be employed for BM cells from rodent strains (rat and mouse). We have also studied the native phenotype by using both immunodetection and immunomagnetic methods and have compared MSCs from mouse, rat and human BM. As is the case for human BM, we have demonstrated that all rodent multipotential CFU-fs are contained within the CD49a-positive cell population. However, in the mouse, the number of CFU-fs is strain-dependent. Interestingly, all rat and mouse Sca-1-positive cells are concentrated within the CD49a-positive fraction and also contain all CFU-fs. In human, the colonies have been detected in the CD49a/CD133 double-positive population. Thus, the CD49a protein is a conserved marker that permits the direct enrichment of BM MSCs from various mammalian species; these cells have been phenotyped as true BM stem cells.