Different Phases Of Differentiation Research Articles

Cell-wall fluorescence highlights the phases of xylogenesis

Summary The monitoring of xylogenesis makes it possible to follow tree growth responses to stress factors in real-time, by observing the course of wood cell division and differentiation. Proper microscopy techniques are of key importance to exactly identify the xylem cells during the different phases of differentiation. We aimed to apply epifluorescence microscopy to follow the lignification process during the different phases of xylogenesis in Mediterranean softwood and hardwood. Microcores from trees of Pinus halepensis Mill. and Arbutus unedo L. were collected at a site in southern Italy, during the period June-December. Fluorescence imaging of sections stained with a water solution of safranin and Astra blue clearly highlighted the contrast between lignified and un-lignified tissue. The proposed methodology is useful to quickly and unambiguously detect the different stages of cell differentiation, as well as the progress in the lignification process. Moreover, it proved to be easily applied to demanding wood materials, such as Mediterranean woods and can be helpful to better track stress responses and the development of anomalies during wood formation, such as intra-annual density fluctuations.

Phox2b Immunohistochemical Staining in Detecting Enteric Neural Crest Cells in Hirschsprung Disease.

It can be challenging to recognize undifferentiated/immature ganglion cells, especially single forms. Ganglion cells and glia are derived from enteric neural crest cells (ENCCs), a group of autonomic nervous system (ANS)-lineage neural crest progenitors that PHOX2B regulates. Phox2b is an excellent marker for neoplastic and non-neoplastic ANS cells (eg, peripheral neuroblastic tumors [pNTs]). We hypothesized that Phox2b immunohistochemical staining (IHC) would also be useful for detecting ENCCs. Hematoxylin and eosin, calretinin IHC, and Phox2b IHC were reviewed on 21 pull-through specimens and on a cohort of 12 rectal biopsies. Phox2b IHC demonstrated nuclear positivity in all of the ganglion cells across the different phases of differentiation without background staining. The Phox2b result correlated with the morphological findings, calretinin IHC results, and diagnoses based on the routine diagnostic method. The intensity was uniformly strong in the undifferentiated/immature forms and became variable in the mature forms; this pattern was similar to that seen in pNTs. Phox2b IHC was highly sensitive and specific for detecting ganglion cells. It worked especially well for immature ganglion cells, seen in premature neonates, and scattered single forms in transition zones. In basic research settings, Phox2b can be a useful marker for early differentiation of ENCCs.

Cancer related fatigue and cancer cachexia are the consequence of endocrine failure caused by persistent stress

This study researches the cause of tumor, cancer related fatigue (CRF) and cancer cachexia (CC), and the relationship among tumor and CRF and CC.Carcinogenesis is consequence of failure of tissue development. Tumor originates from tissue regeneration. The tumor cell is normal incomplete differentiated cell that stop in different phases of differentiation. Tumor promoter stimulates stem cell to proliferate. Carcinogen obstructs stem cell to differentiate. With tumor promoter and carcinogen, the tissue stem cells proliferate but cannot differentiate into mature cell, and form tumors. The disorder of biological signals cell proliferation and differentiation facilitates tumor development.CRF and CC are consequence of endocrine hypofunction and failure caused by persistent stress. Nature factors and psychological factors stimulate organic stress. The significant change of stress is the activation of endocrine system. The persistent stress exhausts the capacity of endocrine glands or hormone receptors of target cells, and leads to endocrine hypofunction even failure. CRF and CC are clinical manifestation of endocrine hypofunction and failure.Cancer is a local lesion, also is a systemic disease. As a local lesion, carcinogen obstructed stem cell to differentiate, developmental failure of local tissue forms tumor. As a systemic disease, cancer is related to natural physical, chemical, and biological factors, as well as negative spiritual factors. The material and spiritual factors induce persistent stress which eventually leads to endocrine hypofunction even failure and unbalance of homeostasis. The disorder of biological signals of cell proliferation and differentiation facilitates tumor development.CRF and CC, as clinical manifestation of endocrine hypofunction and failure, have nothing to do with tumor size and type, but facilitate tumor development. CRF and CC are through all the course of systemic cancerous disease, and commonly precede tumorigenesis. Many patients have been found tumors because of symptoms of CRF and CC. Even if no tumor be found at that time, various tumors would be found in the follow-up.For systemic cancerous disease, cachexia is the cause of death. Most cancer patients do not die of tumor, but of cancer cachexia. Eradicating tumor cell cannot cure systemic cancerous disease; on the contrary, the poisonous side effect of therapies usually speeds up the progress of CC and death. It is important for curing cancer cachexia and restoring the patient's constitution to prevent systemic cancer and improve the quality of life and prolong the survival.

Carcinogenesis is consequence of failure of tissue development

Cancer has become a public health problem. The exploration of pathogenesis and therapy of cancer is mainly under the guidance of gene mutation theory. But the therapeutic effect of cancer is not satisfactory, and many predictions of gene mutation theory do not conform actual phenomena of cancer. The research results of mechanism of genetic molecular mutation trap us in an intricate molecular maze hopelessly. The dilemma compels us to doubt about the correctness of gene mutation theory and re-understand the nature of tumor. This study explores the nature of cancer by the method of theoretical analysis by the view of tissue regeneration, and draws a conclusion that the carcinogenesis is consequence of failure of tissue development. Tumors originate from tissue regeneration, tumor cells originate from normal tissue stem cells. The tumor cells are only normal immature cells. Tumor promoters stimulate stem cells to proliferate. Carcinogens obstruct the inducers from inducing tissue stem cells differentiation outside of cells. With tumor promoters and carcinogens, the tissue stem cells proliferate, but cannot differentiate into mature cells, and stop in different phases of differentiation forming atypical hyperplasia of different degrees and tumors of various differentiation grades in tissue. The tumor cells can differentiate into normal mature cells with suitable inducers. The normal stem cells will develop into tumor stem cells without suitable inducers. The ultimate solution of tumor is differentiation therapy. Stem cell transplantation should be cautious.

Cell viability assessed in a reproducible model of human osteoblasts derived from human adipose-derived stem cells.

Human adipose tissue-derived stem cells (hASCs) have been subjected to extensive investigation because of their self-renewal properties and potential to restore damaged tissues. In the literature, there are several protocols for differentiating hASCs into osteoblasts, but there is no report on the control of cell viability during this process. In this study, we used osteoblasts derived from hASCs of patients undergoing abdominoplasty. The cells were observed at the beginning and end of bone matrix formation, and the expression of proteins involved in this process, including alkaline phosphatase and osteocalcin, was assessed. RANKL, Osterix, Runx2, Collagen3A1, Osteopontin and BSP expression levels were analyzed using real-time PCR, in addition to a quantitative assessment of protein levels of the markers CD45, CD105, STRO-1, and Nanog, using immunofluorescence. Rhodamine (Rho123), cytochrome-c, caspase-3, P-27, cyclin D1, and autophagy cell markers were analyzed by flow cytometry to demonstrate potential cellular activity and the absence of apoptotic and tumor cell processes before and after cell differentiation. The formation of bone matrix, along with calcium nodules, was observed after 16 days of osteoinduction. The gene expression levels of RANKL, Osterix, Runx2, Collagen3A1, Osteopontin, BSP and alkaline phosphatase activity were also elevated after 16 days of osteoinduction, whereas the level of osteocalcin was higher after 21 days of osteoinduction. Our data also showed that the cells had a high mitochondrial membrane potential and a low expression of apoptotic and tumor markers, both before and after differentiation. Cells were viable after the different phases of differentiation. This proposed methodology, using markers to evaluate cell viability, is therefore successful in assessing different phases of stem cell isolation and differentiation.

Conditioned media from differentiating craniofacial bone marrow stromal cells influence mineralization and proliferation in periodontal ligament stem cells.

Previous reports have mainly focused on the behavioral responses of human periodontal ligament stem cells (hPDLSCs) in interaction with tibia bone marrow stromal cells (BMSCs). However, there is little study on the biologic features of hPDLSCs under the induction of maxilla BMSCs (M-BMSCs) at different phases of osteogenic differentiation. We hypothesized that M-BMSCs undergoing osteogenic differentiation acted on the proliferation, differentiation, and bone-forming capacity of hPDLSCs. In this paper, primary hPDLSCs and human M-BMSCs (hM-BMSCs) were expanded in vitro. After screening of surface markers for characterization, hPDLSCs were cocultured with different phases of differentiating hM-BMSCs. Cell proliferation and alkaline phosphatase activity were examined, and mineralization-associated markers such as osteocalcin and runt-related transcription factor 2 of hPDLSCs in coculture with uninduced/osteoinduced hM-BMSCs were evaluated. hPDLSCs in hM-BMSCs-conditioned medium (hM-BMSCs-CM) group showed a reduction in proliferation compared with untreated hPDLSCs, while osteoinduced hM-BMSCs for 10day-conditioned medium (hM-BMSCs-CM-10ds) and osteoinduced hM-BMSCs for 15day-conditioned medium (hM-BMSCs-CM-15ds) enhance the proliferation of hPDLSCs. hM-BMSCs of separate differentiation stages temporarily inhibited osteogenesis of hPDLSCs in the early days. Upon extending time periods, uninduced/osteoinduced hM-BMSCs markedly enhanced osteogenesis of hPDLSCs to different degrees. The transplantation results showed hM-BMSCs-CM-15ds treatment promoted tissue regeneration to generate cementum/periodontal ligament-like structure characterized by hard-tissue formation. This research supported the notion that hM-BMSCs triggered osteogenesis of hPDLSCs suggesting important implications for periodontal engineering.

Differentiation of the zebrafish enteric nervous system and intestinal smooth muscle

Development of the enteric nervous system is critical for normal functioning of the digestive system. In vertebrates, enteric precursors originate from the neural crest and migrate into the digestive system. Enteric neurons enable the digestive system to sense and respond to local conditions without the need for central nervous system input. Here we describe major steps in differentiation of the zebrafish enteric nervous system. During migration and neural differentiation of enteric precursors, we identify regions of the enteric nervous system in different phases of differentiation. Early in migration, a small group of anterior enteric neurons are first to form. This is followed by an anterior to posterior wave of enteric neural differentiation later in the migratory phase. Enteric precursors continue proliferating and differentiating into the third day of embryogenesis. nNOS neurons form early while serotonin neurons form late toward the end of enteric neural differentiation. Numbers of enteric neurons increase gradually except during periods of circular and longitudinal intestinal smooth muscle differentiation.

Effect of leptin in proliferating and differentiated HC11 mouse mammary cells

Leptin and its receptors have been shown to be expressed in several tissues thus suggesting that this protein might be effective not only at the CNS level, but also peripherically. We demonstrated by RT-PCR analysis that leptin and its long isoform receptor are expressed in the mouse mammary epithelial cell line HC11, an in vitro cell model considered suitable to study the regulation of the functional development of the mammary epithelium. Furthermore, leptin secretion by HC11 cells was demonstrated by heterologous ELISA. Neither mRNA expression nor protein secretion changed throughout the different phases of differentiation of the cell line. Receptor mRNA was not modified when cells were induced to express β-casein. High concentrations of leptin (between 1.5 and 15 μM) significantly ( p<0.05) reduced cell growth as measured by MTT test. HC11 cells were transfected with pβcCAT, a chimeric rat-β casein gene promoter-CAT gene construct and CAT ELISA was used to determine gene expression. Leptin, from 1.5 nM to 15 μM, was shown to positively ( p<0.05) influence β-casein expression both in the presence or in the absence of prolactin. These data provide evidence that leptin, through its receptor, may be an important mediator in regulating mammary gland growth and development.

Differential expressions of protein kinase C isozymes during proliferation and differentiation of human skeletal muscle cells in vitro.

The mechanism of skeletal muscle regeneration in vivo can be well modeled in vitro by culturing skeletal muscle cells. In these cultures mononuclear satellite cells fuse to form polynuclear myotubes by proliferation and differentiation. The aim of this study was to determine how the different protein kinase C (PKC) isozymes were expressed during differentiation of human skeletal muscle in vitro. The expressions of desmin, used as a muscle-specific intermediate filament protein marker of differentiation, and of different PKC isozymes were detected by single and double immunohistochemical labeling, and by Western blot analysis. In skeletal muscle cells we could identify five PKC isozymes (PKC alpha, -gamma, -etha, -theta and -zeta). The expressions of PKC alpha and -zeta did not change significantly during differentiation; their levels of expression were high in the early immature cells and remained unchanged in later phases. In contrast, the expression levels of PKC gamma and -etha increased with differentiation. Furthermore, the cellular localization of PKC gamma markedly altered during differentiation, with a perinuclear-nuclear to cytoplasmic translocation. The change in the level of expression of PKC theta during differentiation showed different pattern; its expression was high during the early phases, but a decreased immunostaining was detected in the matured, well-differentiated myotubes. We conclude, therefore, that cultured human skeletal muscle cells possess a characteristic PKC isozyme pattern, and that the different phases of differentiation are accompanied by different expression patterns of the various isozymes. These data suggest the possible functional and differential roles of PKC isozymes in human skeletal muscle differentiation.

Temporal and spatial analysis of hyaluronidase activity during development of the embryonic chick limb bud

The glycosaminoglycan hyaluronate (HA) appears to play an important role in limb cartilage differentiation. The large amount of extracellular HA accumulated by prechondrogenic mesenchymal cells may prevent the cell-cell and/or cell-matrix interactions necessary to trigger chondrogenesis, and the removal of extracellular HA may be essential to initiate the crucial cellular condensation process that triggers cartilage differentiation. It has generally been assumed that HA turnover during chondrogenesis is controlled by the activity of the enzyme hyaluronidase (HAase). In the present study we have performed a temporal and spatial analysis of HAase activity during the progression of limb development and cartilage differentiation in vivo. We have separated embryonic chick wing buds at several stages of development into well-defined regions along the proximodistal axis in which cells are in different phases of differentiation, and we have examined HAase activity in each region. We have found that HAase activity is clearly detectable in undifferentiated wing buds at stage 18 19 , which is shortly following the formation of a morphologically distinct limb bud rudiment, and remains relatively constant throughout subsequent stages of development through stage 27 28 , at which time well-differentiated cartilage rudiments are present. Moreover, HAase activity in the prechondrogenic distal subridge regions of the limb at stages 22 23 and 25 is just as high as, or even slightly higher than, it is in proximal central core regions where condensation and cartilage differentiation are progressing. We have also found that limb bud HAase is active between pH 2.2 and 4.5 and is inactive above pH 5.0. This suggests that limb HAase is a lysosomal enzyme and that extracellular HA would have to be internalized to be degraded. These results indicate that the onset of chondrogenesis is not associated with the appearance or increase in activity of HAase. We suggest the possibility that HA turnover may be regulated by the binding and endocytosis of extracellular HA in preparation for its intracellular degradation by lysosomal HAase. Finally, we have found that the apical ectodermal ridge (AER)-containing distal limb bud ectoderm possesses a relatively high HAase activity. We suggest the possibility that a high HAase activity in the AER may ensure a rapid turnover and remodeling of the disorganized HA-rich basal lamina of the AER that might be essential for limb outgrowth.

Lymphocyte subpopulations in immunodeficiency disorders: I. Human thymus-lymphoid tissue antigen

Peripheral blood lymphocytes from 25 normal persons and 47 patients with immunodeficiency diseases were studied for the presence of human thymus-lymphoid tissue antigen (HTLA) and their responsiveness to PHA. Whereas in normal persons, HTLA values were consistently around 36%, 17 immunodeficient patients had low numbers of HTLA-positive lymphocytes in their peripheral blood. In general, HTLA values corresponded with PHA responsiveness ( 36 47 ), however, a number of discordant situations were observed. These results suggest that HTLA may be a useful marker in evaluating an immunologically relevant lymphocyte population in clinical situations. This population is most likely of thymus origin (4,5) and appears to be associated with PHA responsiveness in most situations. On the other hand, the dissociation of these markers in 11 patients suggests that they detect either distinct but overlapping populations, different phases of differentiation of a single lymphocyte population or that the cells involved may be perturbed independently by exogenous factors, such as infections.

Changes of the acriflavine sensitivity of lymphoid cells of mice pretreated with acriflavine diazotate combined with heterologous albumin

The possibility that the «in vivo» treatment with heterologous albumin coupled with diazotized acriflavine may affect the sensitivity of lymphoid cells to the action of acriflavine was studied. Albino mice CFW strain were treated subcutanceusly with the coupled albumin in the presence of complete Freund adjuvant. Lymph nodes from control and immunized animals, fifteen days after the treament, were cultured «in vitro» in the presence of different doses of acriflavine (from 0.5 to 4 μg/ml). The action of acriflavine was evaluated as the growth of cultures, the percent of lymphoid cells in the different phases of differentiation and the percent of proliferating cells after incubation for 24 hours in the presence of 3H thymidine. Results show that lymphoid cells of immunized mice are less sensitive to the citotoxic activity of acriflavine than those of the controls. Acriflavine, at low doses, reduces the growth of normal cultures and the proliferative activity of immature elements. At the highest doses the proliferation area is almost completely absent and the elements still present are strongly degenerated. Acriflavine, at the concentration able to reduce or to inhibit the growth of control cultures, is ineffective in altering the ratio of immature elements in cultures of immunized animals. The ability of these elements to incorporate 3H thymidine is also unchanged.