低氧对CD34^＋造血干／祖细胞增殖分化及其对细胞因子依赖性的影响

To elucidate the effects of hypoxia on the proliferation and differentiation of CD34(+) hematopoietic stem/progenitor cells and their response to cytokines, the cells were isolated from umbilical cord blood by using a high-gradient magnetic cell sorting system (MACS). Mononuclear cells (MNC) and CD34(+) cells were incubated in severe hypoxia (1% oxygen) culture system, and the colony forming cells and antigen expression were studied by colony forming assays and FACS analysis. The results showed that incubation in severe hypoxia increased the number of erythroid bursts (BFU-E) (324.8+/-41.4/10(4) cells) generated from CD34(+) cells (191.2+/-34.5/10(4) cells in the control group, P<0.01). Severe hypoxia also enhanced the maintenance and cloning efficiency of BFU-E in a liquid culture system without growth factors, the number of BFU-E (152.4+/-22.6/10(4)cells) was much bigger than that in the control group (74.2+/-9.3/10(4) cells, P<0.01). In cultures incubated in hypoxia, the percentage of CD34(+) cells was significantly higher (2.5+/-1.2-fold, P<0.05) than in those incubated in air. BFU-E cloning efficiency of MNC was not significantly affected by hypoxia. The above results show that hypoxia enhances the maintenance of erythroid progenitor cells generated from CD34(+) hematopoietic stem/progenitor cells, no matter growth factors are present or not. These positive effects of hypoxia did not occur for the other progenitors.     

低氧对CD34~ 造血干/祖细胞增殖分化及其对细胞因子依赖性的影响

采用免疫磁珠法分离脐血CD34 造血干 /祖细胞 ,进行低氧和常氧条件下单个核细胞 (MNC)及CD34 细胞的半固体及液体培养 ,计细胞总数和集落产率 ,并通过流式细胞仪检测细胞表型和细胞周期 ,以探讨造血干/祖细胞在低氧环境下增殖分化性能的改变及其对细胞因子反应性的变化。结果显示 :CD34 细胞在低氧条件下生成的BFU E集落数 ( 32 4 8± 41 4/10 4 细胞 )明显增多 (对照为 191 2± 34 5 /10 4 细胞 ,P <0 0 1) ;在无细胞因子存在的液体培养体系中 ,低氧组的BFU E产率 ( 15 2 4± 2 2 6 /10 4 细胞 )明显高于常氧组 ( 74 2± 9 3/10 4 细胞 ,P <0 0 1) ;低氧培养细胞中CD34 细胞的比例高于对照 2 5± 1 2倍 (P <0 0 5 )。但MNC生成的BFU E在常氧和低氧条件下无显著差异。这些结果表明 :体外低氧环境能显著增加CD34 造血干 /祖细胞形成红系祖细胞的产率 ,且使其对细胞因子的依赖性降低 ,并对早期红系祖细胞的维持有增强作用 ,但对粒系祖细胞的增殖则有抑制作用     

Effect of substrate stiffness on proliferation and differentiation of periodontal ligament stem cells

Objectives

The aim of this study was to understand the effect of substrate stiffness (a mechanical factor of the extracellular matrix) on periodontal ligament stem cells (PDLSCs) and its underlying mechanism.

Materials and methods

Elastic substrates were fabricated by mixing 2 components, a base and curing agent in proportions of 10:1, 20:1, 30:1 or 40:1. PDLSC morphology was observed using scanning electron microscopy (SEM). Cell proliferation and differentiation were assessed after PDLSCs was cultured on various elastic substrates. Data were analysed using one‐way ANOVA.

Results

SEM revealed variations in the morphology of PDLSCs cultured on elastic substrates. PDLSC proliferation increased with substrate stiffness (P < .05). Osteogenic differentiation of PDLSCs was higher on stiff substrates. Notch pathway markers were up‐regulated in PDLSCs cultured on stiff substrates.

Conclusions

Results suggested that the osteogenic differentiation of PDLSCs might be promoted by culturing them in a stiffness‐dependent manner, which regulates the Notch pathway. This might provide a new method of enhancing osteogenesis in PDLSCs.

     

In vivo stimulatory effect of macrophage colony-stimulating factor on the number of stroma-initiating cells

The in vivo effect of human macrophage colony-stimulating factor (M-CSF) on the number of cells that formed stromal colonies in an in vitro culture system (stroma-initiating cells; SICs) was investigated. We found that the number of SICs in the femurs of C57BL/6 mice was significantly increased by the treatment with M-CSF. We also found that the SICs were resistant to at least three different chemotherapeutic reagents, 5-fluorouracil (5-FU), cytarabine, and cyclophosphamide, because the femoral cells of mice treated with these reagents contained higher numbers of SICs than those of untreated mice. M-CSF treatment also increased the number of SICs of the reagent-pretreated mice. The SICs detected in our culture system were present only in Mac-1(-)CD45(-) cells, and the M-CSF treatment of 5-FU-pretreated mice actually increased the number of Mac-1(-)CD45(-) SICs. The Mac-1(-)CD45(-) SICs collected from mice that were pretreated with 5-FU and then treated with M-CSF formed stromal colonies under in vitro culture conditions that did not contain M-CSF but did contain a high concentration of fetal calf serum. This result suggested that SICs collected following the treatment procedure did not necessarily require the presence of M-CSF for their in vitro proliferation. Our study indicated that M-CSF has the ability to increase the number of progenitor or precursor cells for bone marrow stromal cells in vivo system.     

Effect of valproic acid and antiapoptotic cytokines on differentiation and apoptosis induction of human leukemia cells

This work compares effect of histondeacetylase inhibitor, valproic acid (VA), on proliferation, differentiation and apoptosis induction in two human leukemic cell lines: HL-60 (human promyleocytic leukemia, p53 negative) and MOLT-4 (human T-lymphocyte leukemia, p53 wild type). Incubation with VA caused decrease in percentage of cells in S phase of cell cycle. The decrease was more intensive in HL-60 cells, where the cells in S phase were absent 6 days after the beginning of incubation with VA (4 mmol/l). 3-day-long incubation of HL-60 cells with 4 mmol/l VA caused differentiation of these cells, marked by increase in CD11b and co-stimulatory/adhesion molecule CD86, and induction of a significant apoptosis. Annexin V positive cells lost the CD11b antigen. 3-day-long incubation of MOLT-4 cells with VA (1-2 mmol/l) inhibited proliferation and decreased percentage of cells in S phase of the cell cycle. 90% of MOLT-4 cells are CD7 positive. This CD7 positivity is not changed during apoptosis induction (detected as Annexin V positivity). On the other hand, CD4 marker expression decreases after incubation with 1-2 mmol/l VA, but during apoptosis induction by 4 mmol/l VA, most of the apoptotic Annexin V positive cells were also CD4 positive. Using a clonogenic survival assay EC(50) for 3-day-long incubation with VA was determined. For HL-60 cells, the established EC(50) was 1.84 mmol/l, for MOLT-4 cells it was 1.76 mmol/l. Ability of VA to induce differentiation in HL-60 cells thus does not affect final cell killing. However, the elimination of the cells was considerably affected by presence of hematopoietic growth factors. 14-day-long incubation of HL-60 cells with VA in conditioned medium (source of IL-3, SCF, G-CSF) caused increase in EC(50) to 4 mmol/l, while in MOLT-4 cells (cultivation without conditioned medium), the EC(50) decreased to 0.63 mmol/l.     

Effect of inflammatory cytokines on human endothelial cell proliferation.

Neovascularization, a common occurrence in chronic inflammatory lesions, requires endothelial cell (EC) proliferation. Because this form of inflammation is often mediated by immunologically generated cytokines, the effects of such cytokines on human umbilical vein EC proliferation in vitro were investigated. Low concentrations of recombinant interferon gamma (rIFN-gamma) (10-100 U/ml), but not a higher concentration (1,000 U/ml), enhanced both basal and endothelial cell growth factor (ECGF)-stimulated EC proliferation. Recombinant interleukin 1 (rIL-1) and recombinant tumor necrosis factor-alpha (rTNF) had minor effects on basal EC proliferation, but significant inhibition was observed in the presence of ECGF. A combination of rIFN-gamma and rTNF induced marked suppression of EC proliferation, which appeared to be due to a cytotoxic effect on the EC, as demonstrated by 51Cr release. In contrast, the combination of rIFN-gamma and rIL-1 had only an additive effect on EC proliferation, with no evidence of cytotoxicity. These results suggest that cytokines have important regulatory roles in local vascular proliferation. These effects varied not only with the individual cytokine, but also with the combination of cytokines used. The most striking effects were 1) the stimulation of proliferation by IFN-gamma at a low concentration and 2) the inhibition by both rIL-1 and rTNF of ECGF-stimulated proliferation.     

Effect of recombinant IL 2 and gamma-IFN on proliferation and differentiation of human B cells

The effects of IL 2 and gamma-IFN on the activation of human B cells was studied with recombinant IL 2 and gamma-IFN. BCDF-responsive B lymphoblastoid cell lines and highly purified human B cells were employed as target B cells. IL 2 or gamma-IFN did not induce any IgG or IgM secretion in the B cell lines CESS and SKW6-CL4, in which IgG and IgM were inducible with conventional T cell factor(s). IL 2 alone did not induce the optimum production of Ig, but did induce proliferation in the SAC-stimulated B cell population. No Leu-1-, Leu-4-, or Leu-7-positive cells were detected in B cell populations that had been stimulated with SAC for 3 days. FACS analysis showed that a portion of the SAC-stimulated B cells (30%) were in the G2 or M stages by IL 2 stimulation. The addition of gamma-IFN together with IL 2 induced IgM and IgG secretion in SAC-stimulated B cells that was comparable with that induced by a conventional T cell factor(s). IL 2 induced proliferation not only in SAC-stimulated B cells but also in an anti-mu-stimulated B cell population. Stimulation of T cell populations with anti-mu and IL 2 did not induce significant proliferation, suggesting the direct effect of IL 2 on B cells. Double staining of anti-mu-stimulated B cells with anti-Ig and anti-Tac antibodies demonstrated that anti-mu stimulation induced an increased expression of Tac antigen on surface Ig-positive B cells. All of these results strongly supported the notion that IL 2 was one of the growth factors for B cells, and gamma-IFN was one of the differentiation factors for B cells.     

Effect of neurotrophins on differentiation, calcification and proliferation in cultures of human pulp cells

Neurotrophins (NTs) are expressed during tooth development. However, little is known about a role of NTs in differentiation of pulp cells into mineralizing cells. In this study, mRNA expressions of hard tissue-related proteins, calcification and proliferation are examined in cultures of human pulp (HP) cells. Nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin (NT)-3 and NT-4/5 increased the mRNA levels of dentin sialophsphoprotein, alkaline phosphatase, osteopontin, type I collagen and bone morphogenetic protein-2 and mineral deposition in cultures of HP cells. The increased levels and manners varied, depending on the concentrations of NTs and hard-tissue related protein tested. On the other hand, only NGF significantly stimulated DNA synthesis in cultures of HP cells. These findings suggest that NTs characteristically regulate hard-tissue related protein expression, calcification and proliferation in pulp cells. NTs may accelerate pulp cell differentiation.     

Effect of angiotensin II on proliferation and differentiation of mouse induced pluripotent stem cells into mesodermal progenitor cells

Previous studies suggest that angiotensin receptor stimulation may enhance not only proliferation but also differentiation of undifferentiated stem/progenitor cells. Therefore, in the present study, we determined the involvement of the angiotensin receptor in the proliferation and differentiation of mouse induced pluripotent stem (iPS) cells. Stimulation with angiotensin II (Ang II) significantly increased DNA synthesis in mouse iPS cells cultured in a medium with leukemia inhibitory factor (LIF). Pretreatment of the cells with either candesartan (a selective Ang II type 1 receptor [AT(1)R] antagonist) or Tempol (a cell-permeable superoxide scavenger) significantly inhibited Ang II-induced DNA synthesis. Treatment with Ang II significantly increased JAK/STAT3 phosphorylation. Pretreatment with candesartan significantly inhibited Ang II- induced JAK/STAT3 phosphorylation. In contrast, induction of mouse iPS cell differentiation into Flk-1-positive mesodermal progenitor cells was performed in type IV collagen (Col IV)- coated dishes in a differentiation medium without LIF. When Col IV-exposed iPS cells were treated with Ang II for 5days, the expression of Flk-1 was significantly increased compared with that in the cells treated with the vehicle alone. Pretreatment of the cells with both candesartan and SB203580 (a p38 MAPK inhibitor) significantly inhibited the Ang II- induced increase in Flk-1 expression. Treatment with Ang II enhanced the phosphorylation of p38 MAPK in Col IV- exposed iPS cells. These results suggest that the stimulation of mouse iPS cells with AT(1)R may enhance LIF-induced DNA synthesis, by augmenting the generation of superoxide and activating JAK/STAT3, and that AT(1)R stimulation may enhance Col IV-induced differentiation into mesodermal progenitor cells via p38 MAPK activation.     

Six family genes control the proliferation and differentiation of muscle satellite cells

Muscle satellite cells are essential for muscle growth and regeneration and their morphology, behavior and gene expression have been extensively studied. However, the mechanisms involved in their proliferation and differentiation remain elusive. Six1 and Six4 proteins were expressed in the nuclei of myofibers of adult mice and the numbers of myoblasts positive for Six1 and Six4 increased during regeneration of skeletal muscles. Six1 and Six4 were expressed in quiescent, activated and differentiated muscle satellite cells isolated from adult skeletal muscle. Overexpression of Six4 and Six5 repressed the proliferation and differentiation of satellite cells. Conversely, knockdown of Six5 resulted in augmented proliferation, and that of Six4 inhibited differentiation. Muscle satellite cells isolated from Six4^+/−Six5^−/− mice proliferated to higher cell density though their differentiation was not altered. Meanwhile, overproduction of Six1 repressed proliferation and promoted differentiation of satellite cells. In addition, Six4 and Six5 repressed, while Six1 activated myogenin expression, suggesting that the differential regulation of myogenin expression is responsible for the differential effects of Six genes. The results indicated the involvement of Six genes in the behavior of satellite cells and identified Six genes as potential target for manipulation of proliferation and differentiation of muscle satellite cells for therapeutic applications.     

In vitro characterization of proliferation and differentiation of pig satellite cells

Skeletal muscle contains various muscle fiber types exhibiting different contractile properties based on the myosin heavy chain (MyHC) isoform profile. Muscle fiber type composition is highly variable and influences growth performance and meat quality, but underlying mechanisms regulating fiber type composition remain poorly understood. The aim of the present work was to develop a model based on muscle satellite cell culture to further investigate the regulation of adult MyHC isoforms expression in pig skeletal muscle. Satellite cells were harvested from the mostly fast-twitch glycolytic longissimus (LM) and predominantly slow-twitch oxidative rhomboideus (RM) muscles of 6-week-old piglets. Satellite cells were allowed to proliferate up to 80% confluence, reached after 7 day of proliferation (D7), and then induced to differentiate. Kinetics of proliferation and differentiation were similar between muscles and more than 95% of the cells were myogenic (desmin positive) at D7 with a fusion index reaching 65±9% after 4 day of differentiation. One-dimensional SDS polyacrylamide gel electrophoresis revealed that satellite cells from both muscles only expressed the embryonic and fetal MyHC isoforms in culture, without any of the adult MyHC isoforms that were expressed in vivo. Interestingly, triiodothyronine (T3) induced de novo expression of adult fast and α-cardiac MyHC in vitro making our culture system a valuable tool to study de novo expression of adult MyHC isoforms and its regulation by intrinsic and/or extrinsic factors.     

Effect of connective tissue growth factor (CCN2/CTGF) on proliferation and differentiation of mouse periodontal ligament-derived cells

Background

CCN2/CTGF is known to be involved in tooth germ development and periodontal tissue remodeling, as well as in mesenchymal tissue development and regeneration. In this present study, we investigated the roles of CCN2/CTGF in the proliferation and differentiation of periodontal ligament cells (murine periodontal ligament-derived cell line: MPL) in vitro.

Results

In cell cultures of MPL, the mRNA expression of the CCN2/CTGF gene was stronger in sparse cultures than in confluent ones and was significantly enhanced by TGF-β. The addition of recombinant CCN2/CTGF (rCCN2) to MPL cultures stimulated DNA synthesis and cell growth in a dose-dependent manner. Moreover, rCCN2 addition also enhanced the mRNA expression of alkaline phosphatase (ALPase), type I collagen, and periostin, the latter of which is considered to be a specific marker of the periosteum and periodontium; whereas it showed little effect on the mRNA expression of typical osteoblastic markers, e.g., osteopontin and osteocalcin. Finally, rCCN2/CTGF also stimulated ALPase activity and collagen synthesis.

Conclusion

These results taken together suggest important roles of CCN2/CTGF in the development and regeneration of periodontal tissue including the periodontal ligament.     

Effect of head activator on proliferation, head-specific determination and differentiation of epithelial cells in hydra

In hydra the differentiation of head-specific ectodermal epithelial cells from multipotent stem cells is a multistep process in which cell cycle progression is regulated at three restriction points. Head activator acts as a positive signal at these restriction points. At the G2/mitosis boundary of epithelial stem cells head activator functions as a mitogen, being necessary for cell division. Subsequently, in or before S phase, head activator acts as determinant to ensure commitment of epithelial cells to head-specific determination. This effect of head activator requires hundredfold-higher concentrations, and may also require longer incubation times, than for cell proliferation. Epithelial cells thus committed to head-specific differentiation become arrested in G2 as a third and last restriction point in the cell cycle. They require disinhibition by decapitation and probably the presence of head activator for final differentiation, which then occurs in G2.     

Effect of pulsed electromagnetic field on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells

Pulsed electromagnetic fields (PEMFs) have been used clinically to slow down osteoporosis and accelerate the healing of bone fractures for many years. The aim of this study is to investigate the effect of PEMFs on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells (BMMSC). PEMF stimulus was administered to BMMSCs for 8 h per day during culture period. The PEMF applied consisted of 4.5 ms bursts repeating at 15 Hz, and each burst contained 20 pulses. Results showed that about 59% and 40% more viable BMMSC cells were obtained in the PEMF‐exposed cultures at 24 h after plating for the seeding density of 1000 and 3000 cells/cm², respectively. Although, based on the kinetic analysis, the growth rates of BMMSC during the exponential growth phase were not significantly affected, 20–60% higher cell densities were achieved during the exponentially expanding stage. Many newly divided cells appeared from 12 to 16 h after the PEMF treatment as revealed by the cell cycle analysis. These results suggest that PEMF exposure could enhance the BMMSC cell proliferation during the exponential phase and it possibly resulted from the shortening of the lag phase. In addition, according to the cytochemical and immunofluorescence analysis performed, the PEMF‐exposed BMMSC showed multi‐lineage differentiation potential similar to the control group. Bioelectromagnetics 30:251–260, 2009. © 2009 Wiley‐Liss, Inc.     

Effect of biodentine coated with emdogain on proliferation and differentiation of human stem cells from the apical papilla

Molecular Biology Reports - This study assessed the effect of Biodentine coated with Emdogain (Biodentine/Emdogain) on proliferation and differentiation of human stem cells from the apical papilla...     

Levamisole regulates the proliferation of murine liver T cells through Kupffer-cell-derived cytokines

 We have previously shown that levamisole increases the cytotoxic, cytostatic, and proliferative activity of murine nonparenchymal liver cells (NPC) in vitro. We have also shown that the nonadherent subpopulation of NPC, which are composed predominantly of T lymphocytes, is very responsive to this agent when administered to mice. Kupffer cells or immigrant macrophages are also responsive to levamisole but to a lesser extent. These findings prompted us to investigate changes in cytokine production by NPC following-treatment of mice with levamisole (25 mg/kg, i.p.), which may help explain the observed alterations in the immune functions of these cells. We found that levamisole treatment of mice causes a threefold increase in production of interferon (IFN) α/β by adherent NPC (more than 80% – 90% Kupffer cells) in vitro. When IFN α/β was added to cultured cells, it decreased the proliferative capacity of liver T cells in a dose-dependent manner. In contrast, the addition of anti-IFNα/β was shown to augment levamisole-induced proliferation of unfractionated NPC and Kupffer cells. NPC production of interleukin 1 (IL-1) and interleukin-6 (IL-6) in vitro was also increased threefold following treatment of mice with levamisole. IL-6 added in vitro to cells significantly augmented levamisole-induced proliferation of liver T cells while anti-IL-6 reduced proliferative activity to control levels. These findings suggested that IFNα/β, IL-6, and IL-1 play important regulatory roles in controlling the proliferative response of murine liver-associated T lymphocytes to levamisole. Finally, the proliferation of bone marrow cells was increased in mice given 5-fluorouracil (5FU). On the other hand, the proliferation of NPC was dramatically suppressed when 5FU was administered. However, the proliferation of these cells was restored when levamisole was given after 5FU. Received: 27 November 1995 / Accepted: 16 October 1996     

Pro-inflammatory cytokines induce odontogenic differentiation of dental pulp-derived stem cells

Postnatal dental pulp stem cells (DPSCs) represent a unique precursor population in the dental pulp, which have multipotential and harbor great potential for tissue engineering purposes. However, for therapy applications, transplanted cells are often exposed to unfavorable conditions such as cytokines released from necrotic or inflammatory cells in injured tissues. It is not clear how stem cells exposed to these conditions changes in their characteristics. In this study, the effects of pro-inflammatory cytokines, such as IL-1 and TNF, on DPSCs were investigated. Cells were treated with IL-1, TNF, or both for 3, 7, and 12 days. The cultures were evaluated for cell proliferation, ALP activity, and real-time PCR. We found that a short treatment (3 days) of pro-inflammatory cytokines induced the odontogenic differentiation of DPSCs. Furthermore, post 3 days treatment with pro-inflammatory cytokines, the cell-scaffold complexes were implanted subcutaneously in mice for 8 weeks. Histological analysis demonstrated that the cultures gave obviously mineralized tissue formation, especially for both IL-1 and TNF applied. These data suggest that IL-1 and TNF produced in the early inflammatory reaction may induce the mineralization of DPSCs.