In search of mammary gland stem cells

Summary The evidence for mammary epithelial stem cells and their phenotypic characteristics in normal and neoplastic development is reviewed. The presence of stem cells in all parts of the mammary parenchyma at all stages of differentiation has been demonstrated by transplantation experiments. The phenotypic characterization of stem cells has been defined by a battery of monospecific antibodies. These studies suggest that a mammary epithelium stem cell compartment exists in the basal layer of the gland as well as in the end bud. Whether these same stem cells are expressed in mammary preneoplasias and neoplasias has not been answered conclusively. Phenotypic markers specific for stem cells and stably expressed in transformed cell populations are needed to follow the fate of stem cells.Dedicated to Professor Stuart Patton on the occassion of his 70th birthday.     

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.     

A rat mammary gland cancer cell with stem cell properties of self-renewal and multi-lineage differentiation

The cancer stem cell hypothesis posits that tumors are derived from a single cancer-initiating cell with stem cell properties. The task of identifying and characterizing cancer-initiating cells with stem cell properties at the single cell level has proven technically difficult because of the scarcity of the cancer stem cells in the tissue of origin and the lack of specific markers for cancer stem cells. Here we show that a single LA7 cell, derived from rat mammary adenocarcinoma has: the ability to serially re-generate mammospheres in long-term non-adherent cultures, the differentiation potential to generate all the cell lineages of the mammary gland and branched duct-like structures that recapitulate morphologically and functionally the ductal–alveolar-like architecture of the mammary tree. The properties of self-renewal, extensive capacity for proliferation, multi-lineage differentiation and the tubular-like structure formation potential suggest that LA7 cells is a cancer stem model system to study the dynamics of tumor formation at the single cell level. Cinzia Cocola, Sveva Sanzone and Simonetta Astigiano have contributed equally to this work.     

Parity-induced mammary epithelial cells are multipotent and express cell surface markers associated with stem cells

Parity-induced mammary epithelial cells (PI-MECs) are defined as a pregnancy hormone-responsive cell population that activates the promoter of late milk protein genes during the second half of pregnancy and lactation. However, unlike their terminally differentiated counterparts, these cells do not undergo programmed cell death during post-lactational remodeling of the gland. We previously demonstrated that upon transplantation into an epithelial-free mammary fat pad, PI-MECs exhibited two important features of multipotent mammary epithelial progenitors: a) self-renewal, and b) contribution to ductal and alveolar morphogenesis. In this new report, we introduce a new method to viably label PI-MECs. Using this methodology, we analyzed the requirement of ovarian hormones for the maintenance of this epithelial subtype in the involuted mammary gland. Furthermore, we examined the expression of putative stem cell markers and found that a portion of GFP-labeled PI-MECs were part of the CD24(+)/CD49f(high) mammary epithelial subtype, which has recently been suggested to contain multipotent stem cells. Subsequently, we demonstrated that isolated PI-MECs were able to form mammospheres in culture, and upon transplantation, these purified epithelial cells were capable of establishing a fully functional mammary gland. These observations suggest that PI-MECs contain multipotent progenitors that are able to self renew and generate diverse epithelial lineages present in the murine mammary gland.     

Epithelial differentiation of human adipose tissue-derived adult stem cells

Adult human stem cells are employed in novel treatments and bio-artificial devices. Recent studies have identified an abundant source of stem cells termed adipose-derived adult stem (ADAS)-cells in the subcutaneous adipose tissue. Under appropriate culture conditions ADAS-cells differentiate to various cell types, including chondrocytes, adipocytes, and smooth muscle cells. Aiming at epithelial differentiation this study investigated the effect of all-trans retinoic acid (ATRA) on human ADAS-cells. ATRA-induced cytokeratin 18 expression in ADAS-cells and nearly abolished vimentin expression as shown by Western blot. In immunofluorescence, the formation of keratin fibers in ATRA-treated ADAS-cells could be observed. The percentage of ADAS-cells being able to undergo epithelial differentiation as quantified by FACS-analysis was above 80%. Inhibition of cell growth by ATRA was shown using DAPI- and MTT-assays. ATRA can differentiate ADAS-cells toward the epithelial lineage. This finding, along with a previously described neural differentiation, shows that ADAS-cells have epithelial potential.     

Identification of superficial zone articular chondrocyte stem/progenitor cells

Identification of progenitor/stem cell populations that differentiate specifically towards superficial zone articular chondrocytes is an unmet challenge for cartilage tissue engineering. Using fluorescence activated cell sorting (FACS) analysis we found a characteristic pattern of "side population" (SP) stem cells identified by the Hoechst 33342 dye. We established micromass cultures from this population of cells and tested their chondrogeneic potential. Control (untreated) cultures were minimally stained for Alcian blue - a marker of chondrogenesis. However, with BMP-7 treatment, Alcian blue staining was increased. Superficial zone protein - a specific marker for articular cartilage superficial zone chondrocytes - increased with BMP-7 and/or TGF-beta1 treatment in SP micromass cultures. Our results demonstrate the presence of stem/progenitor cells in the SP fraction isolated from the surface zone of bovine cartilage and have the ability to specifically differentiate towards the superficial zone articular chondrocyte.     

Successful immortalization of mesenchymal progenitor cells derived from human placenta and the differentiation abilities of immortalized cells

We reported previously that mesenchymal progenitor cells derived from chorionic villi of the human placenta could differentiate into osteoblasts, adipocytes, and chondrocytes under proper induction conditions and that these cells should be useful for allogeneic regenerative medicine, including cartilage tissue engineering. However, similar to human mesenchymal stem cells (hMSCs), though these placental cells can be isolated easily, they are difficult to study in detail because of their limited life span in vitro. To overcome this problem, we attempted to prolong the life span of human placenta-derived mesenchymal cells (hPDMCs) by modifying hTERT and Bmi-1, and investigated whether these modified hPDMCs retained their differentiation capability and multipotency. Our results indicated that the combination of hTERT and Bmi-1 was highly efficient in prolonging the life span of hPDMCs with differentiation capability to osteogenic, adipogenic, and chondrogenic cells in vitro. Clonal cell lines with directional differentiation ability were established from the immortalized parental hPDMC/hTERT+Bmi-1. Interestingly, hPDMC/Bmi-1 showed extended proliferation after long-term growth arrest and telomerase was activated in the immortal hPDMC/Bmi-1 cells. However, the differentiation potential was lost in these cells. This study reports a method to extend the life span of hPDMCs with hTERT and Bmi-1 that should become a useful tool for the study of mesenchymal stem cells.     

Neuropharmacological properties of neurons derived from human stem cells

Human pluripotent stem cells have enormous potential value in neuropharmacology and drug discovery yet there is little data on the major classes and properties of receptors and ion channels expressed by neurons derived from these stem cells. Recent studies in this lab have therefore used conventional patch-clamp electrophysiology to investigate the pharmacological properties of the ligand and voltage-gated ion channels in neurons derived and maintained in vitro from the human stem cell (hSC) line, TERA2.cl.SP12.TERA2.cl.SP12 stem cells were differentiated with retinoic acid and used in electrophysiological experiments 28-50 days after beginning differentiation. HSC-derived neurons generated large whole cell currents with depolarizing voltage steps (−80 to 30 mV) comprised of an inward, rapidly inactivating component and a delayed, slowly deactivating outward component. The fast inward current was blocked by the sodium channel blocker tetrodotoxin (0.1 μM) and the outward currents were significantly reduced by tetraethylammonium ions (TEA, 5 mM) consistent with the presence of functional Na and K ion channels. Application of the inhibitory neurotransmitters, GABA (0.1-1000 μM) or glycine (0.1-1000 μM) evoked concentration dependent currents. The GABA currents were inhibited by the convulsants, picrotoxin (10 μM) and bicuculline (3 μM), potentiated by the NSAID mefenamic acid (10-100 μM), the general anaesthetic pentobarbital (100 μM), the neurosteroid allopregnanolone and the anxiolytics chlordiazepoxide (10 μM) and diazepam (10 μM) all consistent with the expression of GABA_A receptors. Responses to glycine were reversibly blocked by strychnine (10 μM) consistent with glycine-gated chloride channels. The excitatory agonists, glutamate (1-1000 μM) and NMDA (1-1000 μM) activated concentration-dependent responses from hSC-derived neurons. Glutamate currents were inhibited by kynurenic acid (1 mM) and NMDA responses were blocked by MgCl₂ (2 mM) in a highly voltage-dependent manner.Together, these findings show that neurons derived from human stem cells develop an array of functional receptors and ion channels with a pharmacological profile in keeping with that described for native neurons. This study therefore provides support for the hypothesis that stem cells may provide a powerful source of human neurons for future neuropharmacological studies.     

Application of mesenchymal stem cells derived from human pluripotent stem cells in regenerative medicine

Mesenchymal stem cells (MSCs) represent the most clinically used stem cells in regenerative medicine. However, due to the disadvantages with primary MSCs, such as limited cell proliferative capacity and rarity in the tissues leading to limited MSCs, gradual loss of differentiation during in vitro expansion reducing the efficacy of MSC application, and variation among donors increasing the uncertainty of MSC efficacy, the clinical application of MSCs has been greatly hampered. MSCs derived from human pluripotent stem cells (hPSC-MSCs) can circumvent these problems associated with primary MSCs. Due to the infinite self-renewal of hPSCs and their differentiation potential towards MSCs, hPSC-MSCs are emerging as an attractive alternative for regenerative medicine. This review summarizes the progress on derivation of MSCs from human pluripotent stem cells, disease modelling and drug screening using hPSC-MSCs, and various applications of hPSC-MSCs in regenerative medicine. In the end, the challenges and concerns with hPSC-MSC applications are also discussed.     

The dynamics of murine mammary stem/progenitor cells

The stem/progenitor cells in the murine mammary gland are a highly dynamic population of cells that are responsible for ductal elongation in puberty, homeostasis maintenance in adult, and lobulo-alveolar genesis during pregnancy. In recent years understanding the epithelial cell hierarchy within the mammary gland is becoming particularly important as these different stem/progenitor cells were perceived to be the cells of origin for various subtypes of breast cancer. Although significant advances have been made in enrichment and isolation of stem/progenitor cells by combinations of antibodies against cell surface proteins together with flow cytometry, and in identification of stem/progenitor cells with multi-lineage differentiation and self-renewal using mammary fat pad reconstitution assay and in vivo genetic labeling technique, a clear understanding of how these different stem/progenitors are orchestrated in the mammary gland is still lacking. Here we discuss the different in vivo and in vitro methods currently available for stem/progenitor identification, their associated caveats, and a possible new hierarchy model to reconcile various putative stem/progenitor cell populations identified by different research groups.     

Effect of lithium chloride on cell proliferation and osteogenic differentiation in stem cells from human exfoliated deciduous teeth

Lithium Chloride (LiCl) has been used as a canonical Wnt pathway activator due to its ability to inhibit a glycogen synthase kinase-3. The aim of the present study was to investigate the effect of LiCl on cell proliferation and osteogenic differentiation in stem cells isolated from human exfoliated deciduous teeth (SHEDs). SHEDs were isolated and cultured in media supplemented with LiCl at 5, 10, or 20 mM. The results demonstrated that LiCl significantly decreased SHEDs colony forming unit ability in a dose dependent manner. LiCl significantly enhanced the percentage of cells in the sub G0 phase, accompanied by a reduction of the percentage of cells in the G1 phase at day 3 and 7 after treatment. Further, LiCl markedly decreased OSX and DMP1 mRNA expression after treating SHEDs in an osteogenic induction medium for 7 days. In addition, no significant difference in alkaline phosphatase enzymatic activity or mineral deposition was found. Together, these results imply that LiCl influences SHEDs behavior.     

Organotypic growth and differentiation of human mammary gland in sponge-gel matrix supported histoculture

Summary Blocks of breast tissue obtained during radical mastectomies from 23 patients with mammary gland carcinomas were used for cultivation in native-state, gel-supported histocultures. We show that the human mammary gland can be successfully maintained in this system so that normal epithelial breast structures proliferate and undergo differentiation for several weeks and a well-developed system of ducts and lobules is formed. Using antibodies to individual keratins 17 and 8 we have shown for the first time that ducts and alveoles developing in vitro undergo differentiation into the lining epithelium and myoepithelium in the same way as mammary gland epithelium in vivo. Growth of epithelial structures in vitro is also accompanied by the development of continuous basal membrane.     

Differentiation of adult rat bone marrow stem cells into epithelial progenitor cells in culture

We have previously obtained monoclonal bone marrow stem cells from adult rats (rMSCs) and induced them into phenotypic neurons. In the present study, we aimed to induce rMSCs into epithelial cells by culturing them onto compartmentalized permeable supports, which have been used for growing a variety of polarized epithelia in culture. Hematoxylin staining showed that after 4 days grown on permeable supports, rMSCs formed an epithelial-like monolayer. Immunofluorescence of the permeably-supported monolayers, but not the rMSCs grown in culture flasks, showed positive signals for epithelial markers, cytokeratin 5 & 8. RT-PCR results also showed the mRNA expression of epithelial sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) as well as tight junction protein ZO-1 in the rMSC-derived monolayers grown on permeable supports but absent from those grown in culture flasks. However, western blot only detected protein expression of ZO-1 but not ENaC nor CFTR. The short-circuit current measurements showed that the rMSC-derived monolayers grown on permeable supports exhibited a trans-monolayer resistance of 30-50 Omega cm(2); however, the monolayers did not respond to activators or blockers of CFTR or ENaC. The results suggest that compartmentalized or polarized culture conditions provide a suitable environment for rMSCs to differentiate into epithelial progenitor cells with tight junction formation; however, this condition is not sufficient for functional expression of epithelial ion channels associated with well-differentiated epithelia.     

Pancreatic exocrine enzyme-producing cell differentiation via embryoid bodies from human embryonic stem cells

Mouse embryonic stem cells (ESCs) can be induced to form pancreatic exocrine enzyme-producing cells in vitro in a stepwise fashion that recapitulates the development in vivo. However, there is no protocol for the differentiation of pancreatic-like cells from human ESCs (hESCs). Based upon the mouse ESC model, we have induced the in vitro formation of pancreatic exocrine enzyme-producing cells from hESCs. The protocol took place in four stages. In Stage 1, embryoid bodies (EBs) were formed from dissociated hESCs and then treated with the growth factor activin A, which promoted the expression of Foxa2 and Sox17 mRNAs, markers of definitive endoderm. In Stage 2, the cells were treated with all-trans retinoic acid which promoted the transition to cells that expressed gut tube endoderm mRNA marker HNF1b. In Stage 3, the cells were treated with fibroblast growth factor 7 (FGF7), which induced expression of Pdx1 typical of pancreatic progenitor cells. In Stage 4, treatment with FGF7, glucagon-like peptide 1, and nicotinamide induced the expression amylase (AMY) mRNA, a marker for mature pancreatic exocrine cells. Immunohistochemical staining showed the expression of AMY protein at the edges of cell clusters. These cells also expressed other exocrine secretory proteins including elastase, carboxypeptidase A, chymotrypsin, and pancreatic lipase in culture. Production of these hESC-derived pancreatic enzyme-producing cells represents a critical step in the study of pancreatic organogenesis and in the development of a renewable source of human pancreatic-like exocrine cells.