过表达Notch1胞内域对c-Kit+骨髓间充质干细胞分化的影响

BACKGROUND: Activation of Notch signaling plays a critical role in stem cell differentiation, and this effect seems to be cell-type dependent. Little is reported on the role of activation of Notch1 signaling in the differentiation of c-Kit+ bone marrow mesenchymal stem cells. OBJECTIVE: To analyze the influence of activation of Notch1 signaling on the differentiation of c-Kit+ bone marrow mesenchymal stem cells. METHODS: The Notch1 intracellular domain (N1-ICD) was obtained from the cDNA library by PCR and cloned into BamHI/AgeI digested adenoviral GV314 plasmid to construct N1-ICD overexpressing shuttle plasmid, and the positive clones were verified by sequencing. N1-ICD shuttle plasmid and helper plasmids pBHGloxΔE1,3 Cre were used to co-transfect HEK293T cells to obtain N1-ICD overexpressing adenoviral particles (N1-ICD-Ad). The c-Kit+ subpopulation were isolated from bone marrow mesenchymal stem cells of the Sprague-Dawley rat femur via magnetic activated cell sorting. After transfection of the c-Kit+ BMSCs with N1-ICD-Ad adenovirus, we assessed the activation of Notch1 signaling and differentiation in c-Kit+ bone marrow mesenchymal stem cells by quantitative RT-PCR and immunofluorescent staining. RESULTS AND CONCLUSION: N1-ICD coding sequence was successfully generated from the cDNA library, and then was cloned into the linearized adenoviral vectors GV314. The resistant clones were verified by sequencing. With the assistance of packaging plasmids, recombinant N1-ICD-Ad adenovirus plasmids were successful packaged in HEK293T cells, and its title was 2×1012 PFU/L. c-Kit+ bone marrow mesenchymal stem cells with the purity of 91.6% were successfully isolated from the bone marrow mesenchymal stem cells of the Sprague-Dawley rat femur. Compared with the blank and negative controls, N1-ICD-Ad infection in the c-Kit+ bone marrow mesenchymal stem cells led to substantial accumulation of N1-ICD in the cytoplasm and nuclei, significantly unregulated expressions of Hes1 (a downstream gene of Notch) and cardiomyocyte differentiation genes Nkx2.5 and cTnT, significantly increased the expression of von Willebrand factor, an endothelial cell differentiation gene, and mildly increased the expression of smooth muscle 22α, a smooth muscle cell differentiation gene. These experimental results indicate that the activation of Notch1 signaling contributes to multi-lineages differentiation of c-Kit+ bone marrow mesenchymal stem cells, and the construction of N1-ICD overexpressing adenoviral vector makes the foundation for further research on the role of Notch1 signaling in stem cell biology.      

Vascular smooth muscle cells for use in vascular tissue engineering obtained by endothelial-to-mesenchymal transdifferentiation (EnMT) on collagen matrices

The discovery of the endothelial progenitor cell (EPC) has led to an intensive research effort into progenitor cell-based tissue engineering of (small-diameter) blood vessels. Herein, EPC are differentiated to vascular endothelial cells and serve as the inner lining of bioartificial vessels. As yet, a reliable source of vascular smooth muscle progenitor cells has not been identified. Currently, smooth muscle cells (SMC) are obtained from vascular tissue biopsies and introduce new vascular pathologies to the patient. However, since SMC are mesenchymal cells, endothelial-to-mesenchymal transdifferentiation (EnMT) may be a novel source of SMC. Here we describe the differentiation of smooth muscle-like cells through EnMT. Human umbilical cord endothelial cells (HUVEC) were cultured either under conditions favoring endothelial cell growth or under conditions favoring mesenchymal differentiation (TGF-beta and PDGF-BB). Expression of smooth muscle protein 22alpha and alpha-smooth muscle actin was induced in HUVEC cultured in mesenchymal differentiation media, whereas hardly any expression of these markers was found on genuine HUVEC. Transdifferentiated endothelial cells lost the ability to prevent thrombin formation in an in vitro coagulation assay, had increased migratory capacity towards PDGF-BB and gained contractile behavior similar to genuine vascular smooth muscle cells. Furthermore, we showed that EnMT could be induced in three-dimensional (3D) collagen sponges. In conclusion, we show that HUVEC can efficiently transdifferentiate into smooth muscle-like cells through endothelial-to-mesenchymal transdifferentiation. Therefore, EnMT might be used in future progenitor cell-based vascular tissue engineering approaches to obtain vascular smooth muscle cells, and circumvent a number of limitations encountered in current vascular tissue engineering strategies.     

The role of Notch and IL-7 signaling in early thymocyte proliferation and differentiation

We have analyzed the roles of Notch and IL-7 signaling in the proliferation and differentiation of mouse progenitor thymocyte subpopulations cultured on Notch delta-like-1 ligand-expressing OP9 stromal cells. Using bulk and limiting dilution cultures, we show that DN1 and DN2 cells require both Notch and IL-7 signaling for efficient proliferation and differentiation into cytoplasmic TCRbeta and surface TCRalpha/beta and TCRgamma/delta expressing T cells. Selection for cytoplasmic TCRbeta-positive cells is dependent on preTalpha expression. Both gamma/delta and alpha/beta TCR expressing T cells arising in culture can be efficiently stimulated by anti-CD3 cross-linking, suggesting that they might be functional. The differentiation of adult, but not fetal, DN1 and DN2 thymocytes into CD4 and/or CD8 expressing cells is inhibited by IL-7. Finally, efficient proliferation and differentiation of DN3 cells requires Notch signaling and preTCR expression, but is independent of IL-7.     

Fibroblast growth factor-1-induced ERK1/2 signaling reciprocally regulates proliferation and smooth muscle cell differentiation of ligament-derived endothelial progenitor cell-like cells

The periodontal ligament (PDL) is a fibrous connective tissue located between the tooth root and the alveolar bone. We previously demonstrated that a single cell-derived culture of primarily cultured PDL fibroblasts has the potential to construct an endothelial cell (EC) marker-positive blood vessel-like structure, suggesting that the fibroblastic lineage cells in ligament tissue could act as the endothelial progenitor cells (EPCs), which regenerate to construct a vascular system around the damaged ligament tissue. Moreover, we showed that EPC-like fibroblasts expressed not only EC markers but also smooth muscle cell (SMC) markers. Generally, an interaction between ECs and SMCs regulates blood vessel development and remodeling, and is required for the formation of a mature and functional vascular network. However, the mechanism underlying the SMC differentiation of the ligament-derived EPC-like fibroblasts remains to be clarified. In this study, we showed that suppression of fibroblast growth factor 1 (FGF-1)-induced extracellular signal-regulated kinase 1/2 (ERK1/2) signaling with the MAPK/ERK kinase (MEK) inhibitor U0126 completely abolished the FGF-1-induced proliferation of the ligament-derived EPC-like fibroblasts. In addition, U0126 treatment of FGF-1-stimulated ligament-derived EPC-like fibroblasts significantly induced the SMC differentiation of the cells. Thus, FGF-1-induced ERK1/2 signaling not only promoted the proliferation of the ligament-derived EPC-like fibroblasts, but also suppressed the SMC differentiation of the cells, suggesting that FGF-1 controls the construction of a vascular network around the ligament tissue by regulating the proliferation and SMC differentiation of the EPC-like cells through ERK-mediated signaling.     

The Notch pathway mediates expansion of a progenitor pool and neuronal differentiation in adult neural progenitor cells after stroke

Molecular mechanisms by which stroke increases neurogenesis have not been fully investigated. Using neural progenitor cells isolated from the subventricular zone (SVZ) of the adult rat subjected to focal cerebral ischemia, we investigated the Notch pathway in regulating proliferation and differentiation of adult neural progenitor cells after stroke. During proliferation of neural progenitor cells, ischemic neural progenitor cells exhibited substantially increased levels of Notch, Notch intracellular domain (NICD), and hairy enhancer of split (Hes) 1, which was associated with a significant increase of proliferating cells. Blockage of the Notch pathway by short interfering ribonucleic acid (siRNA) against Notch or a γ secretase inhibitor significantly reduced Notch, NICD and Hes1 expression and cell proliferation induced by stroke. During differentiation of neural progenitor cells, Notch and Hes1 expression was downregulated in ischemic neural progenitor cells, which was coincident with a significant increase of neuronal population. Inhibition of the Notch pathway with a γ secretase inhibitor further substantially increased neurons, but did not alter astrocyte population in ischemic neural progenitor cells. These data suggest that the Notch signaling pathway mediates adult SVZ neural progenitor cell proliferation and differentiation after stroke.     

Relationship between Delta-like and proneural bHLH genes during chick retinal development.

Notch signaling in the retina maintains a pool of progenitor cells throughout retinogenesis. However, two Notch-ligands from the Delta-like gene family, Dll1 and Dll4, are present in the developing retina. To understand their relationship, we characterized Dll1 and Dll4 expression with respect to proliferating progenitor cells and newborn neurons in the chick retina. Dll4 matched the pattern of neural differentiation. By contrast, Dll1 was primarily expressed in progenitor cells. We compared Dll1 and Dll4 kinetic profiles with that of the transiently up-regulated cascade of proneural basic helix-loop-helix (bHLH) genes after synchronized progenitor cell differentiation, which suggested a potential role for Ascl1 in the regulation of Delta-like genes. Gain-of-function assays demonstrate that Ascl1 does influence Delta-like gene expression and Notch signaling activity. These data suggest that multiple sources of Notch signaling from newborn neurons and progenitors themselves coordinate retinal histogenesis.     

Fibroblast growth factor 9 signaling inhibits airway smooth muscle differentiation in mouse lung

In mammalian lungs, airway smooth muscle cells (airway SMCs) are present in the proximal lung adjacent to bronchi and bronchioles, but are absent in the distal lung adjacent to terminal sacs that expand during gas exchange. Evidence suggests that this distribution is essential for the formation of a functional respiratory tree, but the underlying genetic mechanism has not been elucidated. In this study, we test the hypothesis that fibroblast growth factor 9 (Fgf9) signaling is essential to restrict SMC differentiation to the proximal lung. We show that loss of Fgf9 or conditional inactivation of Fgf receptors (Fgfr) 1 and 2 in mouse lung mesenchyme results in ectopic SMCs. Our data support a model where FGF9 maintains a SMC progenitor population by suppressing differentiation and promoting growth. This model also represents our findings on the genetic relationship between FGF9 and sonic hedgehog (SHH) in the establishment of airway SMC pattern. Developmental Dynamics 238:123–137, 2009. © 2008 Wiley‐Liss, Inc.     

Temporal effects of Notch signaling and potential cooperation with multiple downstream effectors on adenohypophysis cell specification in zebrafish

The adenohypophysis (AH) consists of six distinct types of hormone‐secreting cells. In zebrafish, although proper differentiation of all AH cell types has been shown to require Notch signaling within a period of 14–16 h postfertilization (hpf), the mechanisms underlying this process remain to be elucidated. Herein, we observed using the Notch inhibitor dibenzazepine (DBZ) that Notch signaling also contributed to AH cell specification beyond 16 hpf. Specification of distinct cell types was perturbed by DBZ treatment for different time frames, suggesting that AH cells are specified by Notch‐dependent and cell‐type‐specific mechanisms. We also found that two hes‐family genes, her4.1 and hey1, were expressed in the developing AH under the influence of Notch signaling. her4.1 knockdown reduced expression of proopiomelanocortin a (pomca), growth hormone (gh), and prolactin, whereas hey1 was responsible only for gh expression. Simultaneous loss of both Her4.1 and Hey1 produced milder phenotypes than that of DBZ‐treated embryos. Moreover, DBZ treatment from 18 hpf led to a significant down‐regulation of both gh and pomca genes only when combined with injection of a subthreshold level of her4.1‐morpholino. These observations suggest that multiple downstream effectors, including Her4.1 and Hey1, mediate Notch signaling during AH cell specification.     

JAG1-Mediated Notch Signaling Regulates Secretory Cell Differentiation of the Human Airway Epithelium

Basal cells (BC) are the stem/progenitor cells of the human airway epithelium capable of differentiating into secretory and ciliated cells. Notch signaling activation increases BC differentiation into secretory cells, but the role of individual Notch ligands in regulating this process in the human airway epithelium is largely unknown. The objective of this study was to define the role of the Notch ligand JAG1 in regulating human BC differentiation. JAG1 over-expression in BC increased secretory cell differentiation, with no effect on ciliated cell differentiation. Conversely, knockdown of JAG1 decreased expression of secretory cell genes. These data demonstrate JAG1-mediated Notch signaling regulates differentiation of BC into secretory cells.     

Smooth muscle stem cells

Vascular smooth muscle cells (SMCs) originate from multiple types of progenitor cells. In the embryo, the most well studied SMC progenitor is the cardiac neural crest stem cell. Smooth muscle differentiation in the neural crest lineage is controlled by a combination of cell intrinsic factors, including Pax3, Tbx1, FoxC1, and serum response factor, interacting with various extrinsic factors in the local environment such as bone morphogenetic proteins (BMPs), Wnts, endothelin (ET)-1, and FGF8. Additional sources of multipotential cells that give rise to vascular SMCs in the embryo include proepicardial cells and possibly endothelial progenitor cells. In the adult, vascular SMCs must continually repair arterial injuries and maintain functional mass in response to changing demands upon the vessel wall. Recent evidence suggests that this is accomplished, in part, by recruiting multipotential vascular progenitors from bone marrow-derived stem cells as well as from less well defined sources within adult tissues themselves. This article will review our current understanding of the origins of vascular SMCs from multipotential stem and progenitor cells in developing as well as adult vasculature.     

Regulation of alpha-smooth muscle actin protein expression in adipose-derived stem cells

The objective of this work was to study the response of adipose-derived stem cells (ASCs) to exogenous biochemical stimulation, and the potential of ASCs to differentiate toward the smooth muscle cell (SMC) lineage. Immunofluorescence staining and Western blot analysis detected protein expression of the early SMC marker alpha-smooth muscle actin (alpha-SMA) in both control and experiment groups. Expression of alpha-SMA in ASCs significantly increased when treated with transforming growth factor-beta1, while alpha-SMA expression only slightly increased in the presence of retinoic acid (RA), beta-mercaptoethanol and ascorbic acid. Treatment with platelet-derived growth factor-BB, RA and dibutyryl-cyclic adenosine monophosphate decreased the expression of alpha-SMA significantly. While beta-mercaptoethanol and ascorbic acid, as well as RA have resulted in increased alpha-SMA expression in marrow-derived mesenchymal stem cells and other progenitor cells, our results demonstrate that these treatments do not significantly increase alpha-SMA expression, indicating that the differentiation potential of ASCs and mesenchymal stem cells may be fundamentally different.     

DLK1 Promotes Neurogenesis of Human and Mouse Pluripotent Stem Cell-Derived Neural Progenitors Via Modulating Notch and BMP Signalling

A better understanding of the control of stem cell maintenance and differentiation fate choice is fundamental to effectively realising the potential of human pluripotent stem cells in disease modelling, drug screening and cell therapy. Dlk1 is a Notch related transmembrane protein that has been reportedly expressed in several neurogenic regions in the developing brain. In this study, we investigated the ability of Dlk1 in modulating the maintenance and differentiation of human and mouse ESC-derived neural progenitors. We found that DLK1, either employed as an extrinsic factor, or via transgene expression, consistently promoted the generation of neurons in both the mouse and human ESC-derived neural progenitors. DLK1 exerts this function by inducing cell cycle exit of the progenitors, as evidenced by an increase in the number of young neurons retaining BrdU labelling and cells expressing the cycling inhibitor P57Kip2. DLK1 antagonised the cell proliferation activity of Notch ligands Delta 1 and Jagged and inhibited Hes1-mediated Notch signaling as demonstrated by a luciferase reporter assay. Interestingly, we found that DLK1 promotes the neurogenic potential of human neural progenitor cells via suppression of Smad activation when they are challenged with BMP. Together, our data demonstrate for the first time a regulatory role for DLK1 in human and mouse neural progenitor differentiation and establish an interaction between DLK1 and Hes1-mediated Notch signaling in these cells. Furthermore, this study identifies DLK1 as a novel modulator of BMP/Smad signalling.