Lineage potential, plasticity and environmental reprogramming of epithelial stem/progenitor cells.

Recent evidence supports and reinforces the concept that environmental cues may reprogramme somatic cells and change their natural fate. In the present review, we concentrate on environmental reprogramming and fate potency of different epithelial cells. These include stratified epithelia, such as the epidermis, hair follicle, cornea and oesophagus, as well as the thymic epithelium, which stands alone among simple and stratified epithelia, and has been shown recently to contain stem cells. In addition, we briefly discuss the pancreas as an example of plasticity of intrinsic progenitors and even differentiated cells. Of relevance, examples of plasticity and fate change characterize pathologies such as oesophageal metaplasia, whose possible cell origin is still debated, but has important implications as a pre-neoplastic event. Although much work remains to be done in order to unravel the full potential and plasticity of epithelial cells, exploitation of this phenomenon has already entered the clinical arena, and might provide new avenues for future cell therapy of these tissues.

Bone regeneration and stem cells.

?  Introduction ?  Bone fracture healing and healing problems ?  Biomaterial scaffolds and tissue engineering in bone formation -  Bone tissue engineering -  Biomaterial scaffolds -  Synthetic scaffolds -  Micro- and nanostructural properties of scaffolds -  Conclusion ?  Mesenchymal stem cells and osteogenesis -  Bone tissue -  Origin of osteoblasts -  Isolation and characterization of bone marrow derived MSC -  In vitro differentiation of MSC into osteoblast lineage cells -  In vivo differentiation of MSC into bone -  Factors and pathways controlling osteoblast differentiation of hMSC -  Defining the relationship between osteoblast and adipocyte differentiation from MSC -  MSC and sex hormones -  Effect of aging on osteoblastogenesis -  Conclusion ?  Embryonic, foetal and adult stem cells in osteogenesis -  Cell-based therapies for bone -  Specific features of bone cells needed to be advantageous for clinical use -  Development of therapeutic biological agents -  Clinical application concerns -  Conclusion ?  Platelet-rich plasma (PRP), growth factors and osteogenesis -  PRP effects in vitro on the cells involved in bone repair -  PRP effects on osteoblasts -  PRP effects on osteoclasts -  PRP effects on endothelial cells -  PRP effects in vivo on experimental animals -  The clinical use of PRP for bone repair -  Non-union -  Distraction osteogenesis -  Spinal fusion -  Foot and ankle surgery -  Total knee arthroplasty -  Odontostomatology and maxillofacial surgery -  Conclusion ?  Molecular control of osteogenesis -  TGF-β signalling -  FGF signalling -  IGF signalling -  PDGF signalling -  MAPK signalling pathway -  Wnt signalling pathway -  Hedgehog signalling -  Notch signalling -  Ephrin signalling -  Transcription factors regulating osteoblast differentiation -  Conclusion ?  Summary This invited review covers research areas of central importance for orthopaedic and maxillofacial bone tissue repair, including normal fracture healing and healing problems, biomaterial scaffolds for tissue engineering, mesenchymal and foetal stem cells, effects of sex steroids on mesenchymal stem cells, use of platelet-rich plasma for tissue repair, osteogenesis and its molecular markers. A variety of cells in addition to stem cells, as well as advances in materials science to meet specific requirements for bone and soft tissue regeneration by addition of bioactive molecules, are discussed.

Primate adult brain cell autotransplantation, a pilot study in asymptomatic MPTP-treated monkeys.

Autologous brain cell transplantation might be useful for repairing lesions and restoring function of the central nervous system. We have demonstrated that adult monkey brain cells, obtained from cortical biopsy and kept in culture for a few weeks, exhibit neural progenitor characteristics that make them useful for brain repair. Following MPTP treatment, primates were dopamine depleted but asymptomatic. Autologous cultured cells were reimplanted into the right caudate nucleus of the donor monkey. Four months after reimplantation, histological analysis by stereology and TH immunolabeling showed that the reimplanted cells successfully survived, bilaterally migrated in the whole striatum, and seemed to have a neuroprotection effect over time. These results may add a new strategy to the field of brain neuroprotection or regeneration and could possibly lead to future clinical applications.

Expression of intercellular adhesion molecule-1 in UVA-irradiated human skin cells in vitro and in vivo.

Ultraviolet A (UVA) radiation represents an important oxidative stress to human skin and certain forms of oxidative stress have been shown to modulate intercellular adhesion molecule-1 (ICAM-1) expression. ICAM-1 has been shown to play an important part in many immune reactions and the perturbations of this molecule by ultraviolet radiation could have implications in many inflammatory responses. An enhancement immunohistochemical method with avidin/biotin was used for analysing the early effects of UVA radiation on human cell cultures and human skin (340-400 nm). Both in vitro and in vivo data show that ICAM-1 staining in epidermal keratinocytes, which was expressed constitutively, decreased in a UVA dose-dependent manner. The decrease was most noted at 3-6 h following UVA radiation with some ICAM-1 staining returning by 48 h post-UVA. ICAM-1 positive staining in the dermis was specific for vascular structures and was increased 24 h after UVA radiation. Cultured dermal fibroblasts exhibited ICAM-1 staining which increased slightly within 6-48 h post-UVA radiation. As epidermal ICAM-1 expression is depleted following UVA radiation and dermal expression increases due to an increase in the vascular structures, ICAM-1 provides a valuable marker following UVA radiation in human skin that can be readily measured in situ.

Neural differentiation and therapeutic potential of adipose tissue derived stem cells.

Neural tissue has historically been regarded as having poor regenerative capacity but recent advances in the growing fields of tissue engineering and regenerative medicine have opened new hopes for the treatment of nerve injuries and neurodegenerative disorders. Adipose tissue has been shown to contain a large quantity of adult stem cells (ASC). These cells can be easily harvested with low associated morbidity and because of their potential to differentiate into multiple cell types, their use has been suggested for a wide variety of therapeutic applications. In this review we examine the evidence indicating that ASC can stimulate nerve regeneration by both undergoing neural differentiation and through the release of a range of growth factors. We also discuss some of the issues that need to be addressed before ASC can be developed as an effective cellular therapy for the treatment of neural tissue disorders.

Plasticity of fetal cartilaginous cells.

Tissue-specific stem cells found in adult tissues can participate in the repair process following injury. However, adult tissues, such as articular cartilage and intervertebral disc, have low regeneration capacity, whereas fetal tissues, such as articular cartilage, show high regeneration ability. The presence of fetal stem cells in fetal cartilaginous tissues and their involvement in the regeneration of fetal cartilage is unknown. The aim of the study was to assess the chondrogenic differentiation and the plasticity of fetal cartilaginous cells. We compared the TGF-β3-induced chondrogenic differentiation of human fetal cells isolated from spine and cartilage tissues to that of human bone marrow stromal cells (BMSC). Stem cell surface markers and adipogenic and osteogenic plasticity of the two fetal cell types were also assessed. TGF-β3 stimulation of fetal cells cultured in high cell density led to the production of aggrecan, type I and II collagens, and variable levels of type X collagen. Although fetal cells showed the same pattern of surface stem cell markers as BMSCs, both type of fetal cells had lower adipogenic and osteogenic differentiation capacity than BMSCs. Fetal cells from femoral head showed higher adipogenic differentiation than fetal cells from spine. These results show that fetal cells are already differentiated cells and may be a good compromise between stem cells and adult tissue cells for a cell-based therapy.

Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.

Bone marrow hematopoietic stem cells (HSCs) are crucial to maintain lifelong production of all blood cells. Although HSCs divide infrequently, it is thought that the entire HSC pool turns over every few weeks, suggesting that HSCs regularly enter and exit cell cycle. Here, we combine flow cytometry with label-retaining assays (BrdU and histone H2B-GFP) to identify a population of dormant mouse HSCs (d-HSCs) within the lin(-)Sca1+cKit+CD150+CD48(-)CD34(-) population. Computational modeling suggests that d-HSCs divide about every 145 days, or five times per lifetime. d-HSCs harbor the vast majority of multilineage long-term self-renewal activity. While they form a silent reservoir of the most potent HSCs during homeostasis, they are efficiently activated to self-renew in response to bone marrow injury or G-CSF stimulation. After re-establishment of homeostasis, activated HSCs return to dormancy, suggesting that HSCs are not stochastically entering the cell cycle but reversibly switch from dormancy to self-renewal under conditions of hematopoietic stress.

Harvest site influences the growth properties of adipose derived stem cells.

The therapeutic potential of adult stem cells may become a relevant option in clinical care in the future. In hand and plastic surgery, cell therapy might be used to enhance nerve regeneration and help surgeons and clinicians to repair debilitating nerve injuries. Adipose-derived stem cells (ASCs) are found in abundant quantities and can be harvested with a low morbidity. In order to define the optimal fat harvest location and detect any potential differences in ASC proliferation properties, we compared biopsies from different anatomical sites (inguinal, flank, pericardiac, omentum, neck) in Sprague-Dawley rats. ASCs were expanded from each biopsy and a proliferation assay using different mitogenic factors, basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) was performed. Our results show that when compared with the pericardiac region, cells isolated from the inguinal, flank, omental and neck regions grow significantly better in growth medium alone. bFGF significantly enhanced the growth rate of ASCs isolated from all regions except the omentum. PDGF had minimal effect on ASC proliferation rate but increases the growth of ASCs from the neck region. Analysis of all the data suggests that ASCs from the neck region may be the ideal stem cell sources for tissue engineering approaches for the regeneration of nervous tissue.

Laser-Ultraviolet-A induced ultra weak photon emission in human skin cells: A biophotonic comparison between keratinocytes and fibroblasts

Photons participate in many atomic and molecular interactions and processes. Recent biophysical research has discovered an ultraweak radiation in biological tissues. It is now recognized that plants, animal and human cells emit this very weak biophotonic emission which can be readily measured with a sensitive photomultiplier system. UVA laser induced biophotonic emission of cultured cells was used in this report with the intention to detect biophysical changes between young and adult fibroblasts as well as between fibroblasts and keratinocytes. With suspension densities ranging from 1-8x106 cells/ml, it was evident that an increase of the UVA-laser-light induced photon emission intensity could be observed in young as well as adult fibroblastic cells. By the use of this method to determine ultraweak light emission, photons in cell suspensions in low volumes (100 mu l) could be detected, in contrast to previous procedures using quantities up to 10 ml. Moreover, the analysis has been further refined by turning off the photomultiplier system electronically during irradiation leading to the first measurements of induced light emission in the cells after less than 10 mu s instead of more than 100 milliseconds. These significant changes lead to an improvement factor up to 106 in comparison to classical detection procedures. In addition, different skin cells as fibroblasts and keratinocytes stemining from the same donor were measured using this new highly sensitive method in order to find new biophysical insight of light pathways. This is important in view to develop new strategies in biophotonics especially for use in alternative therapies.

hTERT methylation is necessary but not sufficient for telomerase activity in colorectal cells

Colorectal cancers exhibit a high telomerase activity, usually correlated with the hypermethylation of the promoter of its hTERT catalytic subunit. Although telomerase is not expressed in normal tissue, certain proliferative somatic cells such as intestinal crypt cells have demonstrated telomerase activity. The aim of this study was to determine whether a correlation exists between telomerase activity, levels of hTERT methylation and telomere length in tumoral and normal colorectal tissues. Tumor, transitional and normal tissues were obtained from 11 patients with a colorectal cancer. After bisulfite modification of genomic DNA, hTERT promoter methylation was analyzed by methylation-sensitive single-strand conformation analysis (MS-SSCA). Telomerase activity and telomere length were measured by a fluorescent-telomeric repeat amplification protocol assay and by Southern blotting, respectively. A significant increase of hTERT methylation and telomerase activity, and a reduction of the mean telomere length were observed in the tumor tissues compared to the transitional and normal mucosa. In the transitional and normal mucosa, telomerase activity was significantly lower than that in tumor tissues, even with high levels of hTERT methylation. Nevertheless, hTERT promoter methylation was not linearly correlated to telomerase activity. These data indicate that hTERT promoter methylation is a necessary event for hTERT expression, as is telomerase activity. However, methylation is not sufficient for hTERT activation, particularly in normal colorectal cells.

PAX5 activates the transcription of the human telomerase reverse transcriptase gene in B cells.

Telomerase is an RNA-dependent DNA polymerase that synthesizes telomeric DNA. Its activity is not detectable in most somatic cells but it is reactivated during tumorigenesis. In most cancers, the combination of hTERT hypermethylation and hypomethylation of a short promoter region is permissive for low-level hTERT transcription. Activated and malignant lymphocytes express high telomerase activity, through a mechanism that seems methylation-independent. The aim of this study was to determine which mechanism is involved in the enhanced expression of hTERT in lymphoid cells. Our data confirm that in B cells, some T cell lymphomas and non-neoplastic lymph nodes, the hTERT promoter is unmethylated. Binding sites for the B cell-specific transcription factor PAX5 were identified downstream of the ATG translational start site through EMSA and ChIP experiments. ChIP assays indicated that the transcriptional activation of hTERT by PAX5 does not involve repression of CTCF binding. In a B cell lymphoma cell line, siRNA-induced knockdown of PAX5 expression repressed hTERT transcription. Moreover, ectopic expression of PAX5 in a telomerase-negative normal fibroblast cell line was found to be sufficient to activate hTERT expression. These data show that activation of hTERT in telomerase-positive B cells is due to a methylation-independent mechanism in which PAX5 plays an important role.

Research resource: the dynamic transcriptional profile of sertoli cells during the progression of spermatogenesis.

Sertoli cells (SCs), the only somatic cells within seminiferous tubules, associate intimately with developing germ cells. They not only provide physical and nutritional support but also secrete factors essential to the complex developmental processes of germ cell proliferation and differentiation. The SC transcriptome must therefore adapt rapidly during the different stages of spermatogenesis. We report comprehensive genome-wide expression profiles of pure populations of SCs isolated at 5 distinct stages of the first wave of mouse spermatogenesis, using RNA sequencing technology. We were able to reconstruct about 13 901 high-confidence, nonredundant coding and noncoding transcripts, characterized by complex alternative splicing patterns with more than 45% comprising novel isoforms of known genes. Interestingly, roughly one-fifth (2939) of these genes exhibited a dynamic expression profile reflecting the evolving role of SCs during the progression of spermatogenesis, with stage-specific expression of genes involved in biological processes such as cell cycle regulation, metabolism and energy production, retinoic acid synthesis, and blood-testis barrier biogenesis. Finally, regulatory network analysis identified the transcription factors endothelial PAS domain-containing protein 1 (EPAS1/Hif2α), aryl hydrocarbon receptor nuclear translocator (ARNT/Hif1β), and signal transducer and activator of transcription 1 (STAT1) as potential master regulators driving the SC transcriptional program. Our results highlight the plastic transcriptional landscape of SCs during the progression of spermatogenesis and provide valuable resources to better understand SC function and spermatogenesis and its related disorders, such as male infertility.

Autocrine Action of IGF2 Regulates Adult β-Cell Mass and Function.

Insulin-like growth factor 2 (IGF2), produced and secreted by adult β-cells, functions as an autocrine activator of the β-cell insulin-like growth factor 1 receptor signaling pathway. Whether this autocrine activity of IGF2 plays a physiological role in β-cell and whole-body physiology is not known. Here, we studied mice with β-cell-specific inactivation of Igf2 (βIGF2KO mice) and assessed β-cell mass and function in aging, pregnancy, and acute induction of insulin resistance. We showed that glucose-stimulated insulin secretion (GSIS) was markedly reduced in old female βIGF2KO mice; glucose tolerance was, however, normal because of increased insulin sensitivity. While on a high-fat diet, both male and female βIGF2KO mice displayed lower GSIS compared with control mice, but reduced β-cell mass was observed only in female βIGF2KO mice. During pregnancy, there was no increase in β-cell proliferation and mass in βIGF2KO mice. Finally, β-cell mass expansion in response to acute induction of insulin resistance was lower in βIGF2KO mice than in control mice. Thus, the autocrine action of IGF2 regulates adult β-cell mass and function to preserve in vivo GSIS in aging and to adapt β-cell mass in response to metabolic stress, pregnancy hormones, and acute induction of insulin resistance.

'Hearts and bones': the ups and downs of 'plasticity' in stem cell biology.

More than a decade ago, 'plasticity' suddenly became a 'fashionable' topic with overemphasized implications for regenerative medicine. The concept of 'plasticity' is supported by old transplantation work, at least for embryonic cells, and metaplasia is a classic example of plasticity observed in patients. Nevertheless, the publication of a series of papers showing rare conversion of a given cell type into another unrelated cell raised the possibility of using any unaffected tissue to create at will new cells to replace a different failing tissue or organ. This resulted in disingenuous interpretations and a reason not to fund anymore research on embryonic stem cells (ESc). Moreover, many papers on plasticity were difficult to reproduce and thus questioned; raising issues about plasticity as a technical artefact or a consequence of rare spontaneous cells fusion. More recently, reprogramming adult differentiated cells to a pluripotent state (iPS) became possible, and later, one type of differentiated cell could be directly reprogrammed into another (e.g. fibroblasts into neurons) without reverting to pluripotency. Although the latter results from different and more robust experimental protocols, these phenomena also exemplify 'plasticity'. In this review, we want to place 'plasticity' in a historical perspective still taking into account ethical and political implications.

Phase I and phase II xenobiotic reactions and metabolism of the food-borne carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in aggregating liver cell cultures.

Aggregating fetal liver cell cultures were tested for their ability to metabolize xenobiotics using ethoxycoumarin-O-deethylase (ECOD), as marker of phase I metabolism, and glutathione S-transferase (GST), as marker for phase II reactions. Significant basal activities, stable over 14 days in culture were measured for both ECOD and GST activities. The prototype cytochrome P450 inducers, 3-methylcholanthrene (3-MC) and phenobarbital (PB), increased ECOD and GST activities reaching an optimum 7 days after culturing, followed by a decline in activity. This decline was partially prevented by 1% dimethyl sulfoxide (DMSO) added chronically to the culture medium. DMSO was also found to induce ECOD activity and to a lesser extent GST activity. Furthermore, it potentiated in a dose-dependent manner the induction of ECOD by PB. The food-borne carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is metabolically transformed through a number of pathways in vivo. It was therefore used to examine the metabolic capacity in fetal and adult liver cell aggregates. Metabolism of MeIQx was mainly through N2-conjugation, resulting in formation of the N2-glucuronide and sulfamate conjugates for non-induced fetal liver cells. These metabolites were also found in large amounts in non-induced adult liver cells. Low levels of cytochrome P450-mediated ring-hydroxylated metabolites were detected in both non-induced fetal and adult liver cells. After induction with arochlor (PCB) or 3-MC, the major pathway was ring-hydroxylation (cytochrome P450 dependent), followed by conjugation to beta-glucuronic or sulfuric acid. The presence of the glucuronide conjugate of N-hydroxy-MeIQx, a mutagenic metabolite, suggested an induction of P450 CYP1A2. The metabolism of MeIQx by liver cell aggregates is very similar to that observed in vivo and suggests that aggregating liver cell cultures are a useful model for in vitro metabolic studies in toxicology.