Expansion and characterization of cells from surgically removed intervertebral disc fragments in xenogen-free medium.

Low back pain due to degeneration of intervertebral disc (IVD) is a major health problem resulting in significant disability as well as adding to the economic burden. Discectomy is a very common procedure done worldwide to relieve this pain. At present all the surgically removed disc tissue is mostly discarded. However, there are reports that state that progenitor cells in the IVD can be grown ex vivo and have the potential to be used for IVD repair and regeneration. We report here that viable cells can be harvested from surgically removed, herniated disc tissue and can be potentially used in cell based therapy. Further, we have successfully replaced xenogenic supplements such as foetal bovine serum with either autologous serum or human platelet lysate for culturing IVD cells from patient's surgically removed disc tissue, without loss of any cell characteristics, including cell surface markers, growth factor secretion in the conditioned medium and osteogenic and chondrogenic differentiation potential in vitro. The present work will not only contribute to overcoming some of the major barriers in carrying out human clinical trials, but also provide a cheap, alternate source of proteins and growth factors for growing IVD cells ex vivo for therapy.

Elevated integrin α6 expression is involved in the occurrence and development of lung adenocarcinoma, and predicts a poor prognosis: a study based on immunohistochemical analysis and bioinformatics.

OBJECTIVE: To study integrin α6 expression in lung adenocarcinoma tissue through comparison with matching adjacent non-cancerous tissues as well as elucidating the correlation between integrin α6 expression with the clinical parameters of lung adenocarcinoma. We also explore the signal pathways associated with integrin α6 up-regulation. METHODS: The clinical data, cancer tissues, and adjacent non-cancerous tissues of 30 patients diagnosed with lung adenocarcinoma were collected from Taizhou Hospital in Zhejiang Province, China, in 2010. The protein levels of integrin α6 were determined by immunohistochemistry methods. mRNA data of 85 lung adenocarcinoma tissues and 14 normal tissues as well as clinical results were collected from GEO30219. We also collected mRNA data of 533 lung adenocarcinoma tissues and 59 normal tissues as well as the clinical results of 522 patients with lung adenocarcinoma from the Cancer Genome Atlas (TCGA) database. The differences in protein and mRNA levels in cancer tissues and non-cancerous tissues were analyzed, and we subsequently investigated the association between integrin α6 expression and key parameters indicating lung adenocarcinoma progression and overall survival rate. Additionally, the possible pathways involved in the up-regulation of integrin α6 were analyzed by GSEA. RESULTS: The protein levels of integrin α6 in lung adenocarcinoma tissues were significantly higher than those in adjacent tissues (p < 0.01), and were positively correlated with the grade and T stage of lung adenocarcinoma (p < 0.05). Patients with low integrin α6 protein levels had higher survival rates (p < 0.05). The analysis of gene chip data from the TCGA database also showed that the integrin α6 mRNA level was significantly correlated with T stage (p < 0.05), overall survival (OS) rate (p < 0.01), and disease-free survival (DFS) rate (p = 0.005). GSEA gene enrichment analysis identified a series of pathways that may be associated with integrin α6 up-regulation, including the AGR, PYK2, ECM, and PTEN pathways. CONCLUSION: Integrin α6 plays an important role in the occurrence and progression of lung adenocarcinoma and may act as a prognostic predictor of lung adenocarcinoma in patients. Based on the results of the present study, integrin α6 may be a potential target gene for the treatment of lung adenocarcinoma.

Dynamic expression of α6 integrin indicates epidermal cell behaviors.

Skin epidermis is a stratified epithelium that composed of interfollicular epidermis (IFE) and hair follicles (HFs). Integrins are cell-cell and cell-matrix adhesive ligands that play important roles in epidermal cell proliferation, migration and differentiation behaviors. Here, we analyzed the expression of both α6 and ß1 integrins. In vitro epidermal cell culture, both α6 and ß1 integrins displayed downregulation upon high Ca2+ induced differentiation. During wound healing (WH), α6 integrin showed dynamic expression, first greatly upregulated in unclosed wounds and then downregulated upon re-epithelialization. Further analysis of different wound regions confirmed α6 integrin significantly increased in migratory cells and migration was coupled with differentiation. However, expression level of ß1 integrin did not show significant correlation with migration. We discovered that α6 integrin directly indicates epidermal cell differentiation and wound directed migration behaviors with its expression level.

BMP-2 induces angiogenesis by provoking integrin α6 expression in human endothelial progenitor cells.

Bone morphogenetic protein-2 (BMP-2) is a multifunctional cytokine, capable of governing several cellular functions, including proliferation, motility, differentiation, and angiogenesis. Circulating endothelial progenitor cells (EPCs) have been shown to facilitate tissue repair, postnatal neovascularization, and tumor associated angiogenesis. Nevertheless, the impact of BMP-2 on angiogenesis of human EPCs has largely remained a mystery. In this study, we found that BMP-2 promoted cell migration and tube formation of EPCs in a concentration-dependent manner, indicating BMP-2 induced in vitro angiogenesis in human EPCs. Furthermore, BMP-2 significantly increased microvessel formation in Matrigel plug assay, and BMP-2 antagonist noggin prevented BMP-2-induced in vivo angiogenesis. Mechanistic investigations showed BMP-2 profoundly induced the expression of Id-1 and integrin α6 as well as EPCs angiogenesis by activating PI3K/Akt and MEK/ERK signaling pathways. Moreover, knockdown of Id-1 and integrin α6 by siRNA transfection obviously attenuated BMP-2-indueced tube formation of EPCs. These results suggest that BMP-2 promotes angiogenesis in human EPCs through the activation of PI3K/Akt, MEK/ERK, and Id-1/integrin α6 signaling cascades. This is the first demonstration that BMP-2 exhibits the angiogenesis property on human EPCs. BMP-2 might serve as the potential therapeutic target for treatment of angiogenesis-related diseases.

MicroRNA-330-5p negatively regulates ITGA5 expression in human colorectal cancer.

Colorectal cancer (CRC), one of the most prevalent malignant cancers, has high rates pf incidence and is the fourth leading cause of cancer-related deaths for both men and women worldwide. MicroRNAs (miRNAs) play critical roles in the development of various types of cancers. miRNA­330-5p has been implicated in the progression of prostate, neuronal and pancreatic cancers by regulating proliferation, migration, invasion and epithelial-mesenchymal transition of cells. The purpose of the present study was to investigate the expression of miR-330-5p in CRC and identify its target gene(s) that may act in CRC tumorigenesis. We found that miR-330-5p expression was significantly lower in CRC tissues than that in adjacent non-tumorous tissues. Furthermore, we identified integrin α5 (ITGA5) as a new target of miR-330-5p and found that it inhibits ITGA5 expression by directly binding to the 3' untranslated region of ITGA5 mRNA. These results suggest that downregulation of miR-330-5p expression may affect CRC development via modulation of ITGA5 expression.

RFX1 maintains testis cord integrity by regulating the expression of Itga6 in male mouse embryos.

Formation and maintenance of testis cords during embryogenesis are essential for establishing testicular structure and function in adults. At least five genes (Wt1, Dhh, Sox8/Sox9, and Dax1) appear to be required for the maintenance of testis cord integrity in mice. Here, we report that RFX1 is specifically expressed in fetal Sertoli cells. Mouse embryos conditionally deficient in Rfx1 (Rfx1(flox/flox) , Amh-Cre) possessed disrupted testis cords, as the basal lamina lining was fragmented or completely absent in some areas of the testes. Spermatogenesis was blocked, leading to complete infertility. Expression of integrin alpha-6 was significantly decreased in Rfx1-deficient testes compared to control testes; indeed, luciferase and chromatin immunoprecipitation assays indicated that RFX1 directly activates transcription of Itga6 (the gene coding for integrin alpha-6). Taken together, RFX1 transcriptionally targets Itga6 in Sertoli cells, thereby, helping maintain the integrity of the basal lamina during testis cord development. Mol. Reprod. Dev. 83: 606-614, 2016. © 2016 Wiley Periodicals, Inc.

Putative stem cell markers in cervical squamous cell carcinoma are correlated with poor clinical outcome.

BACKGROUND: The aim of this study was to elucidate the value of putative cancer stem cell markers Musashi-1, ALDH1, Sox2, and CD49f in predicting the prognosis in cervical squamous cell carcinoma (CSCC). METHODS: Real-time PCR and immunohistochemistry staining was performed to examine Musashi-1, ALDH1, Sox2, and CD49f expression in archived specimens of CSCC patients with postoperative chemotherapy. Kaplan-Meier analysis and Cox proportional hazards model were used to assess the prognostic impact of CSC markers for overall survival (OS) and recurrent-free survival (RFS). RESULTS: The Real-time PCR data showed that the expression of all markers were increased in CSCC tissues compared with in paired normal cervical tissues (P < 0.05). The IHC result showed that high expression of Msi1, ALDH1, Sox2, and CD49f was found in 25.7%, 43.0%, 62.0% and 29.0% CSCC samples, respectively. Moreover, high expression of Msi1 (P = 0.033 and P = 0.003, respectively), ALDH1 (P = 0.015 and P = 0.002, respectively), and Sox2 (P = 0.005 and P = 0.003, respectively), and low expression of CD49f (P = 0.027 and P = 0.025, respectively) were correlated with poor OS and PFS in CSCC patients. Interestingly, tumors with Msi1(high)/CD49f(low) expression had the poorest prognosis according to Msi1/CD49f stratification. In multivariate Cox regression analysis, Sox2 expression (P = 0.047 and P = 0.018, respectively), ALDH1 expression (P = 0.013 and P = 0.003, respectively), and CD49f expression (P = 0.008 and P = 0.003, respectively) were independent prognostic markers for both OS and RFS. CONCLUSIONS: Our results suggest that cancer stem cell markers are linked with poor prognosis of CSCC patients.

CD49f Acts as an Inflammation Sensor to Regulate Differentiation, Adhesion, and Migration of Human Mesenchymal Stem Cells.

The advent of mesenchymal stem cell (MSC)-based therapies has been an exciting innovation for the treatment of degenerative and inflammatory diseases. However, the surface markers that accurately reflect the self-renewal and differentiation potential of MSCs and their sensitivity to environmental cues remain poorly defined. Here, we studied the role of CD49f in bone marrow MSCs (BMSCs) and the mechanism by which it regulates the behavior of BMSCs under inflammatory conditions. We found that CD49f is preferentially expressed in fetal cells rather than adult cells, CD49f-positive BMSCs possess higher CFU-F formation ability and differentiation potential than CD49f negative cells, and the CD49f expression of BMSCs gradually decreases during in vitro passaging. CD49f knockdown dramatically decreased the differentiation of BMSCs and isoform A was demonstrated to be the main functional form that enhanced the differentiation ability of BMSCs. The influences of inflammatory cytokines on BMSCs revealed that TNF-α downregulated CD49f in BMSCs with impaired differentiation, decreased adhesion to laminins, and increased migration. Moreover, tissue transglutaminase was found to work together with CD49f to regulate the behavior of BMSCs. Finally, we showed that mTOR signaling rather than NF-κB activation mediated CD49f downregulation induced by TNF-α and maintained CD49f homeostasis in BMSCs. Our findings suggest that CD49f is a stemness marker of BMSCs and is tightly correlated with the behavioral changes of BMSCs under inflammatory conditions. These data demonstrate a novel role for CD49f in sensing inflammation through mTOR pathway to further modulate the behavior of MSCs to fulfill the requirements of the body.

Comparative characterization of stem cell marker expression, metabolic activity and resistance to doxorubicin in adherent and spheroid cells derived from the canine prostate adenocarcinoma cell line CT1258.

BACKGROUND: Canine prostate cancer represents a spontaneous animal model for the human counterpart. Cells with stem cell-like character are considered to play a major role in therapeutic resistance and tumor relapse. Thus, the identification of markers allowing for recognition and characterization of these cells is essential. MATERIALS AND METHODS: Expression of 12 stem cell marker genes in the canine prostate cancer cell line CT1258 and spheroid cells generated from these was analyzed by quantitative real-time PCR. In CT1258 and the generated spheroid cells, CD44 and CD133 expression was analyzed by flow cytometry, as well as proliferation and doxorubicin resistance. RESULTS: Integrin alpha-6 (ITGA6) expression and metabolic activity were significantly up-regulated in CT1258-derived spheroid cells, while doxorubicin resistance remained comparable. CONCLUSION: ITGA6 de-regulation and metabolic activity appear to be characteristic of the generated spheres, indicating potential intervention targets.

miR-25 Modulates Invasiveness and Dissemination of Human Prostate Cancer Cells via Regulation of αv- and α6-Integrin Expression.

Altered microRNA (miRNA; miR) expression is associated with tumor formation and progression of various solid cancers. A major challenge in miRNA expression profiling of bulk tumors is represented by the heterogeneity of the subpopulations of cells that constitute the organ, as well as the tumor tissue. Here, we analyzed the expression of miRNAs in a subpopulation of epithelial stem/progenitor-like cells in human prostate cancer [prostate cancer stem cell (PCSC)] and compared their expression profile to more differentiated cancer cells. In both cell lines and clinical prostate cancer specimens, we identified that miR-25 expression in PCSCs was low/absent and steadily increased during their differentiation into cells with a luminal epithelial phenotype. Functional studies revealed that overexpression of miR-25 in prostate cancer cell lines and selected subpopulation of highly metastatic and tumorigenic cells (ALDH(high)) strongly affected the invasive cytoskeleton, causing reduced migration in vitro and metastasis via attenuation of extravasation in vivo. Here, we show, for the first time, that miR-25 can act as a tumor suppressor in highly metastatic PCSCs by direct functional interaction with the 3'-untranslated regions of proinvasive αv- and α6-integrins. Taken together, our observations suggest that miR-25 is a key regulator of invasiveness in human prostate cancer through its direct interactions with αv- and α6-integrin expression.

Tie2-dependent deletion of α6 integrin subunit in mice reduces tumor growth and angiogenesis.

The α6 integrin subunit (α6) has been implicated in cancer cell migration and in the progression of several malignancies, but its role in tumor angiogenesis is unclear. In mice, anti-α6 blocking antibodies reduce tumor angiogenesis, whereas Tie1-dependent α6 gene deletion enhances neovessel formation in melanoma and lung carcinoma. To clarify the discrepancy in these results we used the cre-lox system to generate a mouse line, α6fl/fl­Tie2Cre(+), with α6 gene deletion specifically in Tie2-lineage cells: endothelial cells, pericytes, subsets of hematopoietic stem cells, and Tie2-expressing monocytes/macrophages (TEMs), known for their proangiogenic properties. Loss of α6 expression in α6fl/fl­Tie2Cre(+) mice reduced tumor growth in a murine B16F10 melanoma model. Immunohistological analysis of the tumors showed that Tie2-dependent α6 gene deletion was associated with reduced tumor vascularization and with reduced infiltration of proangiogenic Tie2-expressing macrophages. These findings demonstrate that α6 integrin subunit plays a major role in tumor angiogenesis and TEM infiltration. Targeting α6 could be used as a strategy to reduce tumor growth.

Boosting and rescuing epidermal superior population from fresh keratinocyte cultures.

Epidermal stem cells (EpSCs) hold great expectations in a regenerative medicine context, but innovative methods that permit to obtain a significant yield of EpSCs or stem-like epidermal cells are still required. We propose a two-step strategy to obtain a superior epidermal stem-like cell fraction among primary keratinocytes (KCs) isolated from adult human skin. The approach is based on the combination of rapid adherence to collagen IV with the rock-associated kinase inhibitor (ROCKi) treatment, and the subsequent immunomagnetic separation of the α6(high)/CD71(dim) cell subset. The combined collagen IV and ROCKi treatment showed not only to enhance cells clonogenic capacity, but also to induce an early epidermal phenotypic signature, along with the diminished expression of late differentiation-associated markers. More importantly, collagen IV and the ROCKi efficiently promoted a synergized effect over α6(high)/CD71(dim) expression, boosting the number of highly proliferative KCs stem-like cells as demonstrated by the expression of ki67. This cell fraction showed a superior ability to generate a 3D stratified epithelium formed by cells with successive differentiation phenotypes. Overall, this strategy indulged the possibility to uncover, among adult KCs, a superior epidermal cell population with stem-like proliferation capacity and early differentiation degree to be used in numerous skin regeneration approaches.

Expression of stem cell markers: OCT4, KIT, ITGA6, and ITGB1 in the male germinal epithelium.

Efforts have been made for the isolation and characterization of human stem spermatogonia (SG) which would be of major interest for fertility preservation in oncologic patients. We evaluated the expression of mammalian SG stem cell markers, KIT, OCT4, integrin alpha 6 (ITGA6), and integrin beta 1 (ITGB1) as possible indicators for the isolation of those cells in humans. Two different types of SG were individually isolated by micromanipulation from testicular biopsies of men with conserved spermatogenesis. Expression of mRNA showed the absence of KIT and ITGB1 markers in SG. By immunocytochemistry (IC), protein expression for KIT and integrins revealed two types of SG populations, negative (type-1) and positive (type-2). By immunohistochemistry (IH), protein expression for KIT and ITGB1 also revealed two kinds of SG populations, negative (SG A-dark) and positive (SG A-pale). Results suggest that in humans it may be possible to obtain pure populations of stem SG by using negative KIT((-))/ITGB1((-)) sorting.

Profiling of normal and malignant breast tissue show CD44high/CD24low phenotype as a predominant stem/progenitor marker when used in combination with Ep-CAM/CD49f markers.

BACKGROUND: Accumulating evidence supports cancer to initiate and develop from a small population of stem-like cells termed as cancer stem cells (CSC). The exact phenotype of CSC and their counterparts in normal mammary gland is not well characterized. In this study our aim was to evaluate the phenotype and function of stem/progenitor cells in normal mammary epithelial cell populations and their malignant counterparts. METHODS: Freshly isolated cells from both normal and malignant human breasts were sorted using 13 widely used stem/progenitor cell markers individually or in combination by multi-parametric (up to 9 colors) cell sorting. The sorted populations were functionally evaluated by their ability to form colonies and mammospheres, in vitro. RESULTS: We have compared, for the first time, the stem/progenitor markers of normal and malignant breasts side-by-side. Amongst all markers tested, we found CD44high/CD24low cell surface marker combination to be the most efficient at selecting normal epithelial progenitors. Further fractionation of CD44high/CD24low positive cells showed that this phenotype selects for luminal progenitors within Ep-CAMhigh/CD49f + cells, and enriches for basal progenitors within Ep-CAM-/low/CD49f + cells. On the other hand, primary breast cancer samples, which were mainly luminal Ep-CAMhigh, had CD44high/CD24low cells among both CD49fneg and CD49f + cancer cell fractions. However, functionally, CSC were predominantly CD49f + proposing the use of CD44high/CD24low in combination with Ep-CAM/CD49f cell surface markers to further enrich for CSC. CONCLUSION: Our study clearly demonstrates that both normal and malignant breast cells with the CD44high/CD24low phenotype have the highest stem/progenitor cell ability when used in combination with Ep-CAM/CD49f reference markers. We believe that this extensive characterization study will help in understanding breast cancer carcinogenesis, heterogeneity and drug resistance.

Transcription factors associated with epithelial-mesenchymal transition and cancer stem cells in the tumor centre and margin of invasive breast cancer.

Although tumor surgery aims for a complete resection respecting tumor-specific safety distance, in many cases the most peripheral part, the invasion front, remains in situ. Tumor cells at the tumor margin lose epithelial properties and acquire features of mesenchymal cells. The process of epithelial-to-mesenchymal transition (EMT) has been suggested to be of prime importance for tissue and vessel invasion. Recently, features of EMT were shown to be linked to cells with tumor-founding capability, so- called cancer stem cells (CSC). In this study we show that transcription factors associated with EMT markers Snail, Slug, Twist and Zeb1 are differentially expressed between normal breast epithelium, ductal carcinoma in situ and invasive breast cancer. Both invasive and in situ carcinoma expressed less Slug and Twist and more Zeb1 compared to normal epithelium. Using fluorescence multi-staining the number of potential CSC among invasive cancer cells varied dramatically depending on the staining combination used (18.5% for CD44(+)/CD24(-) and 2.4% for CD49f(+)/CD24(+)). Interestingly, neither transcription factors associated with EMT nor potential CSC counts varied between tumor centre and invasion front. No association of these features with clinical outcome was detected. Our results suggest that reliable in situ markers for EMT are missing for invasive breast cancer. Alternatively, the process of EMT might be activated in tumor cells at the margin as well as the centre. Furthermore, our data show that the bio-markers of CSC detect very variable cell populations within breast cancer, challenging the assumption of a hierarchical organization of CSC in these tumors.

Receptor activator of NF-κB ligand promotes proliferation of a putative mammary stem cell unique to the lactating epithelium.

In mice, CD49f(hi) mammary stem cells (MaSCs) asymmetrically divide to generate CD49f(+) committed progenitor cells that differentiate into CD49f(-) phenotypes of the milk-secreting tissue at the onset of pregnancy. We show CD49f(+) primary mammary epithelial cells (PMECs) isolated from lactating tissue uniquely respond to pregnancy-associated hormones (PAH) compared with CD49f(+) cells from nonlactating tissue. Differentiation of CD49f(+) PMEC in extracellular matrix produces CD49f(-) luminal cells to form differentiated alveoli. The PAH prolactin and placental lactogen specifically stimulate division of CD49f(-) luminal cells, while receptor activator of nuclear factor (NF)-κB ligand (RANKL) specifically stimulates division of basal CD49f(+) cells. In nondifferentiating conditions, we observed a greater proportion of multipotent self-renewing cells, and RANKL treatment activated the RANK pathway in these cultures. Furthermore, we observed the deposition of calcium nodules in a proportion of these cells. These data imply that a MaSC unique to the lactating breast exists in humans, which generates progeny with discrete lineages and distinct response to PAH.

P-cadherin is coexpressed with CD44 and CD49f and mediates stem cell properties in basal-like breast cancer.

Although the luminal progenitor cell of the normal mammary gland hierarchy has been proposed as the cell-of-origin for basal-like breast cancers, finding the cancer stem cell (CSC) phenotype for this malignancy has proven a difficult task, mostly due to the lack of specific markers. Recently, basal-like sporadic and familial cases of breast cancer have been linked to BRCA1 gene inactivation, which enables the upregulation of the target-repressed CDH3/P-cadherin gene, an important biomarker of basal-like breast carcinomas. Previously, we demonstrated that P-cadherin overexpression can mediate aggressive behavior in these tumors. Thus, our aim was to test whether P-cadherin mediates stem cell properties in basal-like breast carcinomas. Using a series of breast cancer cell lines and primary tumors, we showed that P-cadherin was directly associated with the expression of the breast stem markers CD44, CD49f, and aldehyde dehydrogenase 1 in the basal subtype. Moreover, cell population enriched for P-cadherin expression comprised increased in vitro mammosphere-forming efficiency and capacity to grow colonies in three-dimensional cultures as well as greater tumorigenicity. Importantly, an association was found with stem-/progenitor-like phenotypes of the breast, including the luminal progenitor population, CD49f(+) CD24(+). Additionally, P-cadherin expression conferred resistance to x-ray-induced cell death, sustaining a role for this molecule in another stem cell property. In summary, we demonstrated, for the first time, that P-cadherin mediates stem cell properties, which could be explored in order to better define the CSC phenotype of basal-like breast tumors and the cell-of-origin of this malignancy.

Expression of a2, a5 and a6 subunits of integrin in de-differentiated NIH3T3 cells by cell-free extract of embryonic stem cells.

Generation of patient specific stem cells is among the ultimate goals in regenerative medicine. Such a cell needs to be functional when it transplants. Interaction between the matrix proteins and integrin adjust many cells' function such as adhesion, migration, cell cycle and self renewal in stem cells. In this study, NIH3T3 cells were dedifferentiated by mouse Embryonic Stem Cell (mESC) extract. The expression of pluripotency markers as well as a2, a5 and a6 integrin subunits were determined. NIH3T3 cells treated with mESC extract showed noticeable changes in cell morphology as early as day 2 post-treatment forming colonies similar to typical mESC morphology by day 8, after three passages. Alkaline phosphatase (ALP) assay and immunocytochemistry staining were performed for the induced reprogrammed cells. The results indicated that these colonies showed the ALP activity and they express Sox2 and Nanog. RT-PCR revealed that the colonies also express Oct3/4. NIH3T3 cells, ESC and reprogrammed cells expressed a2 integrin. a5 integrin expression was greatest in reprogrammed cells followed by the expression of this integrin in NIH3T3 which in turn was more than in ESC. a6A integrin was expressed in NIH3T3 cells while a6B integrin was expressed in ESC and in very low quantity was expressed in reprogrammed cells. These data provide evidence for both the generation of ES like cells from differentiated somatic cells and the expression profile of integrins after de-differentiation by mESC extract.

CD49f enhances multipotency and maintains stemness through the direct regulation of OCT4 and SOX2.

CD49f (integrin subunit α6) regulates signaling pathways in a variety of cellular activities. However, the role of CD49f in regulating the differentiation and pluripotency of stem cells has not been fully investigated. Therefore, in this study, human mesenchymal stem cells (hMSCs) were induced to form spheres under nonadherent culture conditions, and we found that the CD49f-positive population was enriched in MSC spheres compared with MSCs in a monolayer. The expression of CD49f regulated the ability of hMSCs to form spheres and was associated with an activation of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway. Furthermore, the forced expression of CD49f modulated the proliferation and differentiation potentials of hMSCs through prolonged activation of PI3K/AKT and suppressed the level of p53. We showed that the pluripotency factors OCT4 and SOX2 were recruited to the putative promoter region of CD49f, indicating that OCT4 and SOX2 play positive roles in the expression of CD49f. Indeed, CD49f expression was upregulated in human embryonic stem cells (hESCs) compared with hMSCs. The elevated level of CD49f expression was significantly decreased upon embryoid body formation in hESCs. In hESCs, the knockdown of CD49f downregulated PI3K/AKT signaling and upregulated the level of p53, inducing differentiation into three germ layers. Taken together, our data suggest that the cell-surface protein CD49f has novel and dynamic roles in regulating the differentiation potential of hMSCs and maintaining pluripotency.

Novel measurements of mammary stem cells in human umbilical cord blood as prospective predictors of breast cancer susceptibility in later life.

BACKGROUND: The size of the breast stem-cell pool could underlie the intrauterine roots of breast cancer. We studied whether breast stem cells exist in umbilical cord blood and if they correlate with hematopoietic stem-cell measurements that have been positively associated with perinatal risk factors for breast cancer. SUBJECTS AND METHODS: We isolated mononuclear cells from umbilical cord blood of 170 singleton full-term pregnancies and determined, by reverse transcription polymerase chain reaction, the presence of genes of putative breast epithelial stem-cell/progenitor markers [including epithelial cell adhesion molecule (EpCAM), CD49f (α6-integrin), CD117 (c-kit receptor), CD24, and CD29 (ß1-integrin)]. By immunocytochemistry, we colocalized protein expressions of EpCAM+CD49f+, CD49f+CD24+, and CD24+CD29+. We correlated concentrations of putative breast stem-cell/progenitor subpopulations, quantified by flow cytometry, with concentrations of hematopoietic stem cells. RESULTS: Mammary stem-cell phenotypes were identified in umbilical cord blood. The measured EpCAM+ subpopulation was positively correlated with concentrations of CD34+ and CD34+CD38- hematopoietic stem cells (both P=0.006). Additionally, EpCAM+CD49f+ and CD49f+CD24+ subpopulations were positively correlated to the CD34+ cells (P=0.03 and 0.008, respectively). CONCLUSION: The positive association between measurable breast and hematopoietic stem cells in human umbilical cord blood suggests plausible mechanisms for a prenatal influence on breast cancer risk.