Development of a multipotent clonal human periodontal ligament cell line

Abstract The periodontal ligament (PDL) that anchors the tooth root to the alveolar bone influences the lifespan of the tooth, and PDL lost through periodontitis is difficult to regenerate. The development of new PDL-regenerative therapies requires the isolation of PDL stem cells. However, their characteristics are unclear due to the absence of somatic PDL stem cell lines and because PDL is composed of heterogeneous cell populations. Recently, we succeeded in immortalizing human PDL fibroblasts that retained the properties of the primary cells. Therefore, we aimed to establish a human PDL-committed stem cell line and investigate the effects of basic fibroblast growth factor (bFGF) on the osteoblastic differentiation of the cells. Here, we report the development of cell line 1–17, a multipotent clonal human PDL cell line that expresses the embryonic stem cell-related pluripotency genes Oct3/4 and Nanog , as well as the PDL-related molecules periostin and scleraxis. Continuous treatment of cell line 1–17 with bFGF in osteoblastic induction medium inhibited its calcification, with down-regulated expression of FGF-Receptor 1 ( FGF-R1 ), whereas later addition of bFGF potentiated its calcification. Furthermore, bFGF induced calcification of cell line 1–17 when it was co-cultured with osteoblastic cells. These results suggest that cell line 1–17 is a PDL-committed stem cell line and that bFGF exerts dualistic (i.e., promoting and inhibitory) effects on the osteoblastic differentiation of cell line 1–17 based on its differentiation stage.     

Ednrb2 orients cell migration towards the dorsolateral neural crest pathway and promotes melanocyte differentiation

Endothelin receptors B (Ednrb) are involved in the development of the enteric and melanocytic lineages, which originate from neural crest cells (NCCs). In mice, trunk NCCs and their derivatives express only one Ednrb. In quail, trunk NCCs express two Ednrb: Ednrb and Ednrb2. Quail Ednrb is expressed in NCCs migrating along the ventral pathway, which gives rise to the peripheral nervous system, including enteric ganglia. Ednrb2 is upregulated in NCCs before these cells enter the dorsolateral pathway. The NCCs migrating along the dorsolateral pathway are melanocyte precursors. We analyzed the in vitro differentiation and in ovo migration of mouse embryonic stem (ES) cells expressing and not expressing Ednrb2. We generated a series of transfected ES cell lines expressing Ednrb2. This receptor, like Ednrb, oriented genuine ES cells towards melanocyte lineage differentiation in vitro. The in ovo migration of Ednrb2-expressing ES cells was massively oriented towards the dorsolateral pathway, unlike that of WT or Ednrb-expressing ES cells. Thus, Ednrb2 is involved in melanoblast differentiation and migration.     

Investigating a clonal human periodontal ligament progenitor/stem cell line in vitro and in vivo

The lifespan of the tooth is influenced by the periodontal ligament (PDL), a specialized connective tissue that connects the cementum with the tooth socket bone. Generation of a cell line from PDL progenitor/stem cells would allow development of tissue engineering-based regenerative PDL therapy. However, little is known about the characteristics of PDL progenitor/stem cells because PDL tissue consists of a heterogeneous cell population and there are no pure PDL cell lines. Recently, we succeeded in immortalizing primary human PDL fibroblasts (HPLFs) by transfecting them with SV40 T-antigen and hTERT (Cell Tissue Res 2006; 324: 117-125). In this study, we isolated three clonal cell lines from these immortalized cells (lines 1-4, 1-11, and 1-24) that express RUNX-2, Col I, ALP, OPN, OCN, RANKL, OPG, scleraxis, periostin, Col XII, and alpha-SMA mRNA. Immunocytochemical analysis demonstrated that CD146 was expressed in cell lines 1-4 and 1-11 and that STRO-1 was expressed in lines 1-11 and 1-24. Lines 1-4 and 1-11 differentiated into osteoblastic cells and adipocytes when cultured in lineage-specific differentiation media. Four weeks after transplanting cell line 1-11 into immunodeficient mice with beta-tricalcium phosphate (beta-TCP), the transplant produced cementum/bone-like tissues around the beta-TCP. Eight weeks after transplantation, the 1-11 cell transplant formed PDL-like structures on the surface of the beta-TCP. These data suggest that cell line 1-11 was derived from a progenitor/stem cell present in the PDL and should be very useful for studying the biology and regeneration of human periodontium.     

Fibronectin promotes differentiation of neural crest progenitors endowed with smooth muscle cell potential

The neural crest (NC) is a model system used to investigate multipotency during vertebrate development. Environmental factors control NC cell fate decisions. Despite the well-known influence of extracellular matrix molecules in NC cell migration, the issue of whether they also influence NC cell differentiation has not been addressed at the single cell level. By analyzing mass and clonal cultures of mouse cephalic and quail trunk NC cells, we show for the first time that fibronectin (FN) promotes differentiation into the smooth muscle cell phenotype without affecting differentiation into glia, neurons, and melanocytes. Time course analysis indicated that the FN-induced effect was not related to massive cell death or proliferation of smooth muscle cells. Finally, by comparing clonal cultures of quail trunk NC cells grown on FN and collagen type IV (CLIV), we found that FN strongly increased both NC cell survival and the proportion of unipotent and oligopotent NC progenitors endowed with smooth muscle potential. In contrast, melanocytic progenitors were prominent in clonogenic NC cells grown on CLIV. Taken together, these results show that FN promotes NC cell differentiation along the smooth muscle lineage, and therefore plays an important role in fate decisions of NC progenitor cells.     

A chemotactic model of trunk neural crest cell migration

Trunk neural crest cells follow a common ventral migratory pathway but are distributed into two distinct locations to form discrete sympathetic and dorsal root ganglia along the vertebrate axis. Although fluorescent cell labeling and time‐lapse studies have recorded complex trunk neural crest cell migratory behaviors, the signals that underlie this dynamic patterning remain unclear. The absence of molecular information has led to a number of mechanistic hypotheses for trunk neural crest cell migration. Here, we review recent data in support of three distinct mechanisms of trunk neural crest cell migration and develop and simulate a computational model based on chemotactic signaling. We show that by integrating the timing and spatial location of multiple chemotactic signals, trunk neural crest cells may be accurately positioned into two distinct targets that correspond to the sympathetic and dorsal root ganglia. In doing so, we honor the contributions of Wilhelm His to his identification of the neural crest and extend the observations of His and others to better understand a complex question in neural crest cell biology.     

Immortalization and controlled in vitro differentiation of murine multipotent neural crest stem cells

To isolate mouse neural crest stem cells, we have generated a rat monoclonal antibody to murine neurotrophin receptor (p75). We have immortalized p75⁺ murine neural crest cells by expression of v-myc, and have isolated several clonal cell lines. These lines can be maintained in an undifferentiated state, or induced to differentiate by changing the culture conditions. One of these cell lines, MONC-1, is capable of generating peripheral neurons, glia, and melanocytic cells. Importantly, most individual MONC-1 cells are multipotent when analyzed at clonal density. The neurons that differentiate under standard conditions have an autonomic-like phenotype, but under different conditions can express markers of other peripheral neuronal lineages. These lines therefore exhibit a similar differentiation potential as their normal counterparts. Furthermore, they can be genetically modified or generated from mice of different genetic backgrounds, providing a useful tool for molecular studies of neural crest development. © 1997 John Wiley & Sons, Inc. J Neuroblol 32 : 722–746, 1997     

Control of neural crest cell behavior and migration

Neural crest cells (NCCs) are a remarkable, dynamic group of cells that travel long distances in the embryo to reach their target sites. They are responsible for the formation of craniofacial bones and cartilage, neurons and glia in the peripheral nervous system, and pigment cells. Live imaging of NCCs as they traverse the embryo has been critical to increasing our knowledge of their biology. NCCs exhibit multiple behaviors and communicate with each other and their environment along each step of their journey. Imaging combined with molecular manipulations has led to insights into the mechanisms controlling these behaviors. In this review, we highlight studies that have used live imaging to provide novel insight into NCC migration and discuss how continued use of such techniques can advance our understanding of NCC biology.     

Generation of a human airway epithelium derived basal cell line with multipotent differentiation capacity

Background

As the multipotent progenitor population of the airway epithelium, human airway basal cells (BC) replenish the specialized differentiated cell populations of the mucociliated airway epithelium during physiological turnover and repair. Cultured primary BC divide a limited number of times before entering a state of replicative senescence, preventing the establishment of long-term replicating cultures of airway BC that maintain their original phenotype.

Methods

To generate an immortalized human airway BC cell line, primary human airway BC obtained by brushing the airway epithelium of healthy nonsmokers were infected with a retrovirus expressing human telomerase (hTERT). The resulting immortalized cell line was then characterized under non-differentiating and differentiating air-liquid interface (ALI) culture conditions using ELISA, TaqMan quantitative PCR, Western analysis, and immunofluorescent and immunohistochemical staining analysis for cell type specific markers. In addition, the ability of the cell line to respond to environmental stimuli under differentiating ALI culture was assessed.

Results

We successfully generated an immortalized human airway BC cell line termed BCi-NS1 via expression of hTERT. A single cell derived clone from the parental BCi-NS1 cells, BCi-NS1.1, retains characteristics of the original primary cells for over 40 passages and demonstrates a multipotent differentiation capacity into secretory (MUC5AC, MUC5B), goblet (TFF3), Clara (CC10) and ciliated (DNAI1, FOXJ1) cells on ALI culture. The cells can respond to external stimuli such as IL-13, resulting in alteration of the normal differentiation process.

Conclusion

Development of immortalized human airway BC that retain multipotent differentiation capacity over long-term culture should be useful in understanding the biology of BC, the response of BC to environmental stress, and as a target for assessment of pharmacologic agents.     

Analysis of neural crest cell lineage and migration.

In this review, we describe the results of recent experiments designed to investigate various aspects of neural crest cell lineage and migration. We have analyzed the lineage of individual premigratory neural crest cells by injecting a fluorescent lineage tracer dye, lysinated fluorescein dextran, into cells within the dorsal neural tube. Individual clones contained cells that were located in very diverse sites consistent with their being sensory neurons, prepigment cells, Schwann cells, adrenergic cells, and neural tube cells. These results suggest that some neural crest cells in the trunk and cranial regions are multipotent prior to their emigration from the neural tube. The environment through which neural crest cells move influences both the pattern and direction of their migration. We have shown that the sclerotomal portion of the somites are responsible for the rostrocaudal pattern of trunk neural crest cell movement, whereas the neural tube appears to govern the dorsoventral position of neural crest-derived ganglia. In addition, the notochord inhibits the movement of neural crest cells. In order to understand necessary cell-matrix interactions in neural crest migration, we have performed perturbation experiments, in which antibodies directed against cell surface or extracellular matrix molecules were introduced along neural crest pathways. We find that integrins, fibronectin, laminin, and tenascin all play some role in cranial neural crest emigration. Thus, multiple factors may be involved in controlling neural crest cell migration, and different factors may be important for migration in different regions of the embryo.     

Co-culture with endothelial progenitor cells promotes survival,migration, and differentiation of osteoclast precursors

In this study, we report the effect of endothelial progenitor cells (EPCs) on the biological behavior of osteoclast precursors in vitro by establishing an indirect co-culture system of mice EPCs and RAW 264.7 monocyte cells. Results show that the survival, migration, and differentiation of osteoclast precursors were greatly enhanced when co-cultured with EPCs. These phenotypic changes coincide with the upregulation of multiple genes affected cell behavior, including phospho-VEGFR-2, CXCR4, phospho-Smad2/3, phospho-Akt, phospho-ERK1, and phospho-p38 MAPK. The results collectively suggest that EPCs could modulate the survival, migration, and differentiation potential of osteoclast precursors, thus providing new insights in understanding of correlation between angiogenesis and bone homeostasis.     

Establishment of an epidermal growth factor-dependent, multipotent neural precursor cell line

Summary We have established a multipotent clonal cell line, named MEB5, from embryonic mouse forebrains after the infection of a retrovirus carrying E7 oncogene of human papillomavirus type 16. MEB5 cells proliferated in serum-free, epidermal growth factor (EGF)-supplemented medium. They expressed markers for neural precursor cells (nestin, A2B5, and RC1) and did not express markers for neurons (class III β-tubulin), astrocytes (glial fibrillary acidic protein), and oligodendrocytes (galactocerebroside). MEB5 cells were stably maintained in an undifferentiated state with a diploid karyotype in the presence of EGF. When they were deprived of EGF, about 50% of the cells died due apoptosis within 24 h. The remaining cells differentiated into neurons, astrocytes, or oligodendrocytes within 2 wk. The newly developed cells with neuronal morphology were immunoreactive for γ-aminobutyric acid and exhibited neuronal electrophysiological properties. When MEB5 cells were treated with leukemia inhibitory for 7 d, they were induced to differentiate exclusively into astrocytes. These results inducate that MEB5 is a cell line with characteristics of EGF-dependent, multipotent neural precursor cells. This cell line should provide a good model system to study the mechanisms of survival, proliferation, and differentiation of the multipotent precursor cells in the central nervous system.     

Endogenous expression of matriptase in neural progenitor cells promotes cell migration and neuron differentiation

Recent studies show that type II transmembrane serine proteases play important roles in diverse cellular activities and pathological processes. Their expression and functions in the central nervous system, however, are largely unexplored. In this study, we show that the expression of one such member, matriptase (MTP), was cell type-restricted and primarily expressed in neural progenitor (NP) cells and neurons. Blocking MTP expression or MTP activity prevented NP cell traverse of reconstituted basement membrane, whereas overexpression of MTP promoted it. The NP cell mobilization induced by either vascular endothelial growth factor or hepatocyte growth factor was also impaired by knocking down MTP expression. MTP acts upstream of matrix metalloproteinase 2 in promoting NP cell mobility. In embryonic stem cell differentiation to neural cells, MTP knockdown had no effect on entry of embryonic stem cells into the neural lineage. High MTP expression or activity, however, shifts the population dynamics from NP cells toward neurons to favor neuronal differentiation. This is the first report to demonstrate the direct involvement of type II transmembrane serine protease in NP cell function.     

In vivo clonal analysis reveals self-renewing and multipotent adult neural stem cell characteristics

Neurogenesis and gliogenesis continue in discrete regions of the adult mammalian brain. A fundamental question remains whether cell genesis occurs from distinct lineage-restricted progenitors or from self-renewing and multipotent neural stem cells in the adult brain. Here, we developed a genetic marking strategy for lineage tracing of individual, quiescent, and nestin-expressing radial glia-like (RGL) precursors in the adult mouse dentate gyrus. Clonal analysis identified multiple modes of RGL activation, including asymmetric and symmetric self-renewal. Long-term lineage tracing in?vivo revealed a significant percentage of clones that contained RGL(s), neurons, and astrocytes, indicating capacity of individual RGLs for both self-renewal and multilineage differentiation. Furthermore, conditional Pten deletion in RGLs initially promotes their activation and symmetric self-renewal but ultimately leads to terminal astrocytic differentiation and RGL depletion in the adult hippocampus. Our study identifies RGLs as self-renewing and multipotent neural stem cells and provides novel insights into in?vivo properties of adult neural stem cells.