Evidence of progenitor cells of glandular and myoepithelial cell lineages in the human adult female breast epithelium: a new progenitor (adult stem) cell concept

Although experimental data clearly confirm the existence of self-renewing mammary stem cells, the characteristics of such progenitor cells have never been satisfactorily defined. Using a double immunofluorescence technique for simultaneous detection of the basal cytokeratin 5, the glandular cytokeratins 8/18 and the myoepithelial differentiation marker smooth muscle actin (SMA), we were able to demonstrate the presence of CK5+ cells in human adult breast epithelium. These cells have the potential to differentiate to either glandular (CK8/18+) or myoepithelial cells (SMA+) through intermediary cells (CK5+ and CK8/18+ or SMA+). We therefore proceeded on the assumption that the CK5+ cells are phenotypically and behaviourally progenitor (committed adult stem) cells of human breast epithelium. Furthermore, we furnish evidence that most of these progenitor cells are located in the luminal epithelium of the ductal lobular tree. Based on data obtained in extensive analyses of proliferative breast disease lesions, we have come to regard usual ductal hyperplasia as a progenitor cell-derived lesion, whereas most breast cancers seem to evolve from differentiated glandular cells. Double immunofluorescence experiments provide a new tool to characterize phenotypically progenitor (adult stem) cells and their progenies. This model has been shown to be of great value for a better understanding not only of normal tissue regeneration but also of proliferative breast disease. Furthermore, this model provides a new tool for unravelling further the regulatory mechanisms that govern normal and pathological cell growth.     

Efficient in vitro generation of adult multipotent cells from mobilized peripheral blood CD133+ cells

Abstract. Objectives : To generate non-haematopoietic tissues from mobilized haematopoietic CD133⁺ stem cells. Materials and methods : Mobilized peripheral blood CD133⁺ cells from adult healthy donors were used. In vitro ability of highly enriched CD133⁺ cells from mobilized peripheral blood to generate multipotent cells, and their potential to give rise to cells with characteristics of neuroectoderm, endoderm and mesoderm layers was investigated. Results : We found that a recently identified population of CD45⁺ adherent cells generated in vitro after culture of highly purified CD133⁺ cells for 3–5 weeks with Flt3/Flk2 ligand and interleukin-6 can, in presence of the appropriate microenvironmental cues, differentiate into neural progenitor-like cells (NPLCs), hepatocyte-like cells and skeletal muscle-like cells. We have termed them to be adult multipotent haematopoietic cells (AMHCs). AMHC-derived NPLCs expressed morphological, phenotypic and molecular markers associated with primary neural progenitor cells. They can differentiate into astrocyte-like cells, neuronal-like cells and oligodendrocyte-like cells. Moreover, AMHC-derived NPLCs produced 3,4-dihydrophenylalanine and dopamine and expressed voltage-activated ion channels, suggesting their functional maturation. In addition, AMHC-derived hepatocyte-like cells and skeletal muscle-like cells, showed typical morphological features and expressed primary tissue-associated proteins. Conclusion : Our data demonstrate that AMHCs may therefore serve as a novel source of adult multipotent cells for autologous replacement cell therapies.     

Multipotent mesenchymal stem cells from adult human eye conjunctiva stromal cells

Abstract Identification of mesenchymal stem cells (MSCs) derived from alternative sources has provided an exciting prospect for intensive investigation. This work focused on characterizing a new source of MSCs from stromal cells from human eye conjunctiva. In this study, after conjunctiva biopsies and culture of stromal segment of this tissue, fibroblast-like (SH2⁺, SH3⁺, CD29⁺, CD44⁺, CD166⁺, CD13⁺) human stromal cells, which can be differentiated toward the osteogenic, adipogenic, chondrogenic, and neurogenic lineages, were obtained. These cells expressed Oct-4, Nanog, Rex-1 genes, and some lineage-specific markers like cardiac actin and Keratin. Taken together, the results indicate that conjunctiva stromal-derived cells are a new source of multipotent MSCs and despite originating from an adult source, they express undifferentiated stem cell markers.     

Crucial role of the local micro-environment in fate decision of neonatal rat NG2 progenitors

Objectives:  The fate choice of neural progenitor cells could be dictated by local cellular environment of the adult CNS. The aim of our study was to investigate the effect of hippocampal tissue on differentiation and maturation of oligodendrocyte NG2 precursor cells.
Materials and methods:  Hippocampal slice culture was established from the brains of 7-day-old rats. NG2 precursor cells, obtained from a 12-day-old mixed primary culture of neonatal rat cerebral hemispheres, were labelled with chloromethyl-fluorescein-diacetete and seeded on the hippocampal slices. After 7–14 days in co-culture, cells were stained with neural markers.
Results:  NG2 cells differentiated predominantly into oligodendrocytes, presenting various stages of maturation: progenitors (NG2), pre-oligodendrocytes (O4) and finally mature GalC-positive cells. However, except for a few cells with astrocyte-specific S100b staining, a considerable number of these cells differentiated into neurons: TUJ⁺ and even MAP-2⁺ cells were frequently observed. Moreover, a certain population of these cells preserved proliferative properties of primary precursor cells, as revealed by Ki67 expression.
Conclusions:  The neuronal micro-environment provided by the culture of hippocampal slices is potent for induction of neurogenesis from oligodendrocyte NG2⁺/PDGFRα⁺/CNP⁺ progenitor cells and promotes their differentiation not only into macroglia but also into neurons. It also sustains their proliferative capacity. The results indicate the crucial role of the local cellular environment in fate decision of primary NG2⁺ multipotent neural progenitor cells, which may affect their behaviour after transplantation into the central nervous system.     

Signalling through TLR2/MyD88 induces differentiation of murine bone marrow stem and progenitor cells to functional phagocytes in response to Candida albicans

We have previously demonstrated that inactivated yeasts and hyphae of Candida albicans induce in vitro the proliferation of murine haematopoietic stem and progenitor cells (HSPCs, sorted as LKS cells: Lin^- c-Kit⁺ Sca-1⁺) as well as their differentiation to lineage-positive cells, through a MyD88-dependent pathway. In this work, we have found that this process is mainly mediated by TLR2, and that expanding cells express myeloid and not lymphoid markers. Incubation of long-term repopulating HSCs (Lin^- CD105⁺ and Sca-1⁺) with C. albicans yeasts resulted in their proliferation and up regulation of the common myeloid progenitors (CMPs) markers, CD34 and FcγRII/III, by a TLR2/MyD88-dependent signalling pathway. In addition, this TLR2/MyD88 signalling promotes the differentiation of CMPs and granulocyte and macrophage progenitors (GMPs) into cells with the morphology of macrophages and neutrophils, characterized by an increase in the expression of CD11b, F4/80 and Ly6G, independently of the presence of growth and differentiation factors. These differentiated cells were able to phagocytose C. albicans yeasts and to produce proinflammatory cytokines. In conclusion, C. albicans may be sensed by TLRs on haematopoietic stem and progenitor cells to promote the host capability for rapidly replenishing myeloid cells that constitute the first line of defence against C. albicans .     

Clonal proliferation of multipotent stem/progenitor cells in the neonatal and adult salivary glands

Salivary gland stem/progenitor cells are thought to be present in intercalated ductal cells, but the fact is unclear. In this study, we sought to clarify if stem/progenitor cells are present in submandibular glands using colony assay, which is one of the stem cell assay methods. Using a low-density culture of submandibular gland cells of neonatal rats, we developed a novel culture system that promotes single cell colony formation. Average doubling time for the colony-forming cells was 24.7 (SD=+/-7.02)h, indicating high proliferative potency. When epidermal growth factor (EGF) and hepatocyte growth factor (HGF) were added to the medium, the number of clonal colonies increased greater than those cultured without growth factors (13.2+/-4.18 vs. 4.5+/-1.73). The RT-PCR and immunostaining demonstrated expressing acinar, ductal, and myoepithelial cell lineage markers. This study demonstrated the presence of the salivary gland stem/progenitor cells that are highly proliferative and multipotent in salivary glands.     

Source of cell injected is a critical factors for short and long engraftment in xeno-transplantation

Abstract. This study aims to investigate engraftment of human cord blood and foetal bone marrow stem cells after in utero transplantation via the intracoelomic route in the sheep. Here, we performed transplantation in 14 single and 1 twin sheep foetuses at 40–47 days of development, using a novel schedule for injection. (i) Single injection of CD34⁺ human cord blood stem cells via the coelomic route (from 10 to 50 × 10⁴) in seven single foetuses. (ii) Single injection of CD34⁺ foetal bone marrow stem cells via the intracoelomic route with further numbers of cells (20 × 10⁵ and 8 × 10⁵, respectively) in three single and in one twin foetuses. (iii) Double fractioned injection (20–30 × 10⁶) via the coelomic route and 20 × 10⁶ postnatally, intravenously, shortly after birth of CD3-depleted cord blood stem cells in four single foetuses. In the first group, three single foetuses showed human/sheep chimaerism at 1, 8 and 14 months after birth. In the second group, the twin foetuses showed human/sheep chimaerism at 1 month after birth. In the third group, only two out of four single foetuses that underwent transplantation showed chimaerism at 1 month. While foetal bone marrow stem cells showed good short-term engraftment (1 month after birth), cord blood stem cells were able to persist longer in the ovine recipients (at 1, 8 and 14 months after birth).     

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.     

Long-term clinical results of autologous infusion of mobilized adult bone marrow derived CD34+ cells in patients with chronic liver disease

Abstract.  Evidence is growing in support of the role of stem cells as an attractive alternative in treatment of liver diseases. Recently, we have demonstrated the feasibility and safety of infusing CD34⁺ adult stem cells; this was performed on five patients with chronic liver disease. Here, we present the results of long-term follow-up of these patients. Between 1 × 10⁶ and 2 × 10⁸ CD34⁺ cells were isolated and injected into the portal vein or hepatic artery. The patients were monitored for side effects, toxicity and changes in clinical, haematological and biochemical parameters; they were followed up for 12–18 months. All patients tolerated the treatment protocol well without any complications or side effects related to the procedure, also there were no side effects noted on long-term follow-up. Four patients showed an initial improvement in serum bilirubin level, which was maintained for up to 6 months. There was marginal increase in serum bilirubin in three of the patients at 12 months, while the fourth patient's serum bilirubin increased only at 18 months post-infusion. Computed tomography scan and serum α-foetoprotein monitoring showed absence of focal lesions. The study indicated that the stem cell product used was safe in the short and over long term, by absence of tumour formation. The investigation also illustrated that the beneficial effect seemed to last for around 12 months. This trial shows that stem cell therapy may have potential as a possible future therapeutic protocol in liver regeneration.     

Derivation of myoepithelial progenitor cells from bipotent mammary stem/progenitor cells

There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Recent molecular profiling has identified six major subtypes of breast cancer: basal-like, ErbB2-overexpressing, normal breast epithelial-like, luminal A and B, and claudin-low subtypes. To help understand the relationship among mammary stem/progenitor cells and breast cancer subtypes, we have recently derived distinct hTERT-immortalized human mammary stem/progenitor cell lines: a K5(+)/K19(-) type, and a K5(+)/K19(+) type. Under specific culture conditions, bipotent K5(+)/K19(-) stem/progenitor cells differentiated into stable clonal populations that were K5(-)/K19(-) and exhibit self-renewal and unipotent myoepithelial differentiation potential in contrast to the parental K5(+)/K19(-) cells which are bipotent. These K5(-)/K19(-) cells function as myoepithelial progenitor cells and constitutively express markers of an epithelial to mesenchymal transition (EMT) and show high invasive and migratory abilities. In addition, these cells express a microarray signature of claudin-low breast cancers. The EMT characteristics of an un-transformed unipotent mammary myoepithelial progenitor cells together with claudin-low signature suggests that the claudin-low breast cancer subtype may arise from myoepithelial lineage committed progenitors. Availability of immortal MPCs should allow a more definitive analysis of their potential to give rise to claudin-low breast cancer subtype and facilitate biological and molecular/biochemical studies of this disease.     

Regeneration of functional alveoli by adult human SOX9+ airway basal cell transplantation

Irreversible destruction of bronchi and alveoli can lead to multiple incurable lung diseases. Identifying lung stem/progenitor cells with regenerative capacity and utilizing them to reconstruct functional tissue is one of the biggest hopes to reverse the damage and cure such diseases. Here we showed that a rare population of SOX9⁺ basal cells (BCs) located at airway epithelium rugae can regenerate adult human lung. Human SOX9⁺ BCs can be readily isolated by bronchoscopic brushing and indefinitely expanded in feeder-free condition. Expanded human SOX9⁺ BCs can give rise to alveolar and bronchiolar epithelium after being transplanted into injured mouse lung, with air-blood exchange system reconstructed and recipient’s lung function improved. Manipulation of lung microenvironment with Pirfenidone to suppress TGF-β signaling could further boost the transplantation efficiency. Moreover, we conducted the first autologous SOX9⁺ BCs transplantation clinical trial in two bronchiectasis patients. Lung tissue repair and pulmonary function enhancement was observed in patients 3–12 months after cell transplantation. Altogether our current work indicated that functional adult human lung structure can be reconstituted by orthotopic transplantation of tissue-specific stem/progenitor cells, which could be translated into a mature regenerative therapeutic strategy in near future.     

INFLUENCE OF AGE AND ANTIGENIC STIMULATION ON GRANULOCYTE AND MACROPHAGE PROGENITOR CELLS IN THE MOUSE SPLEEN

The frequency and proliferative activity of granulocytic and macrophage progenitor cells were determined in the spleens of C₅₇BL, BALD/c, NZB and CBA mice. These cells were detected by their capacity to form granulocytic and/or macrophage colonies ( in vitro colony-forming cells, CFC) in agar culture. In vitro CFCs were low in frequency in the adult spleen (4–28/10⁵ cells) compared with the bone marrow (180–280/10⁵ cells). However, the neonatal spleen, both in germfree and conventional mice, contained high levels of in vitro CFCs. From the low suiciding index with tritiated thymidine and the small numbers of cluster-forming cells in relation to colony numbers, many in vitro CFCs in the adult C₅₇BL spleen appear to be in a non-cycling state. The level and activity of in vitro CFCs were extremely low in the spleen of adult germfree CBA mice but were greatly increased in conventional mice following the injection of a bacterial antigen.     

OK-432 and IL-2-augmental cytotoxicity of human natural killer cells and cytotoxic T lymphocytes at the clonal level

Abstract The biology response modifiers OK-432 and interleukin-2 (IL-2) were found to enhance the lytic capacity of cloned CD3⁻ natural killer (NK) cells and CD3⁺ T cells. With respect to NK cells, only those clones with a high proliferative capacity and cultured without phytohaemagglutinin (PHA) responded with enhaced lytic capacity to OK-432. OK-432, but not IL-2, was found to augment the antibody-dependent cellular cytotoxicity of cloned NK cells. With T-cell clones, OK-432 augmented the cellular cytotoxicity of CD3⁺8⁺ but not that of CD3⁺4⁺ cytotoxic T lymphocytes, while IL-2 augmented the cytotoxicity of both types of clone. Taken together, these results indicate that OK-432-augmented lytic capacity is not restricted to NK cells and its pathway of action may be independent of IL-2.     

Co-culture with mesenchymal stromal cells increases proliferation and maintenance of haematopoietic progenitor cells

Mesenchymal stromal cells (MSC) have been suggested to provide a suitable cellular environment for in vitro expansion of haematopoietic stem and progenitor cells (HPC) from umbilical cord blood. In this study, we have simultaneously analysed the cell division history and immunophenotypic differentiation of HPC by using cell division tracking with carboxyfluorescein diacetate N -succinimidyl ester (CFSE). Co-culture with MSC greatly enhanced proliferation of human HPC, especially of the more primitive CD34⁺CD38⁻ fraction. Without co-culture CD34 and CD133 expressions decreased after several cell divisions, whereas CD38 expression was up-regulated after some cell divisions and then diminished in fast proliferating cells. Co-culture with MSC maintained a primitive immunophenotype (CD34⁺, CD133⁺ and CD38⁻) for more population doublings, whereas up-regulation of differentiation markers (CD13, CD45 and CD56) in HPC was delayed to higher numbers of cell divisions. Especially MSC of early cell passages maintained CD34 expression in HPC over more cell divisions, whereas MSC of higher passages further enhanced their proliferation rate. Inhibition of mitogen-activated protein kinase 1 (MAPK1) impaired proliferation and differentiation of HPC, but not maintenance of long-term culture initiating cells. siRNA knockdown of N-cadherin and VCAM1 in feeder layer cells increased the fraction of slow dividing HPC, whereas knockdown of integrin beta 1 (ITGB1) and CD44 impaired their differentiation. In conclusion, MSC support proliferation as well as self-renewal of HPC with primitive immunophenotype. The use of early passages of MSC and genetic manipulation of proteins involved in HPC–MSC interaction might further enhance cord blood expansion on MSC.     

Prospective isolation and characterization of mesenchymal stem cells from human placenta using a frizzled-9-specific monoclonal antibody

Abstract We have recently shown that frizzled-9 (FZD9, CD349) is expressed on the cell surface of cultured mesenchymal stromal cells (MSC) derived from the human bone marrow (BM) and chorionic placenta (PL). To study whether FZD9 is also a marker for naive mesenchymal stem cells (MSC), we analyzed the expression pattern of FZD9 on freshly isolated PL cells and determined the clonogenic potential of isolated FZD9⁺ cells using the colony-forming units-fibroblastic (CFU-F) assay. About 0.2% of isolated PL cells were positive for FZD9. Two-color analysis revealed that FZD9⁺ PL cells uniformly express CD9, CD63, and CD90, but are heterogeneous for CD10, CD13, and CD26 expression. In contrast to BM-derived MSC, PL-derived MSC expressed only low levels of CD271. Colony assays of sorted cells showed that clonogenic CFU-F reside exclusively in the FZD9⁺ but not in the FZD9⁻ fraction. Further analysis revealed that CFU-F were enriched by 60-fold in the FZD9⁺CD10⁺CD26⁺ fraction but were absent in the FZD9⁺CD10⁻CD26⁻ population. Cultured FZD9⁺ cells expressed the embryonic stem cell makers Oct-4 and nanog as well as SSEA-4 and TRA1-2-49/6E. In addition, they could be differentiated into functional adipocytes and osteoblasts. This report describes for the first time that FZD9 is a novel and specific marker for the prospective isolation of MSC from human term PL.     

Change of specific T cells in an emerging neonatal infectious disease induced by a bacterial superantigen

A new epidemic, NTED, has recently occurred in Japan. The cause of NTED is a bacterial superantigen, TSST-1. The aim of the present study was to analyze the change in Vβ2⁺ T cells reactive to TSST-1 in NTED in order to establish T-cell-targeted diagnostic criteria for NTED. Blood samples from 75 patients with clinically diagnosed NTED were collected from 13 neonatal intensive care units throughout Japan. We investigated the percentages of Vβ2⁺, Vβ3⁺ and Vβ12⁺ T cells and their CD45RO expressions in the samples using flow cytometry. In 18 of the 75 patients, we conducted multiple examinations of the T cells and monitored serial changes. The Vβ2⁺ T-cell population rapidly changed over three phases of the disease. Whereas the percentage of Vβ2⁺ T cells was widely distributed over the entire control range, CD45RO expression on Vβ2⁺ T cells in CD4⁺ in all 75 patients was consistently higher than the control range. Patients cannot necessarily be diagnosed as having NTED based on expansion of Vβ2⁺ T cells alone in the early acute phase. Instead, CD45RO expression on specific Vβ2⁺ cells is a potential diagnostic marker for a rapid diagnosis of NTED. We present three diagnostic categories of NTED. Fifty patients (66.7%) were included in the category 'definitive NTED'. It is important to demonstrate an increase of Vβ2⁺ T cells in the following phase in cases of 'probable NTED' or 'possible NTED'.     

Vaccination with SIVmac239Δnef activates CD4+ T cells in the absence of CD4+ T-cell loss

Background  Pathogenic HIV and SIV infections characteristically deplete central memory CD4⁺ T cells and induce chronic immune activation, but it is controversial whether this also occurs after vaccination with attenuated SIVs and whether depletion or activation of CD4⁺ T-cell play roles in protection against wild-type virus challenge.
Methods  Rhesus macaques were vaccinated with SIVmac239Δnef and quantitative and phenotypic polychromatic flow cytometry analyses were performed on mononuclear cells from blood, lymph nodes and rectal biopsies.
Results  Animals vaccinated with SIVmac239Δnef demonstrated no loss of CD4⁺ T cells in any tissue, and in fact CCR5⁺ and CD28⁺CD95⁺ central memory CD4⁺ T cells were significantly increased. In contrast, CD4⁺ T-cell numbers and CCR5 expression significantly declined in unvaccinated controls challenged with SIVmac239. Also, intracellular Ki67 increased acutely as much as 3-fold over baseline in all tissues after SIVmac239Δnef vaccination then declined following primary infection.
Conclusion  We demonstrated in this study that SIVmac239Δnef vaccination did not deplete CD4⁺ T cells but transiently activated and expanded the memory cell population. However, increases in numbers and activation of memory CD4⁺ T cells did not appear to influence protective immunity.