Li Z.,Key Laboratory for Regenerative Medicine |
Li Z.,Chinese University |
Li Z.,Jinan University |
Li Z.,Chinese University of Hong Kong |
And 18 more authors.
Journal of Clinical Rehabilitative Tissue Engineering Research | Year: 2011
BACKGROUND: Adult bone marrow-derived stem cells are promising cell source in cell therapy. Tracking of adult bone marrow-derived stem cells is crucial to demonstrate the mechanism of stem cell migration and differentiation, and develop novel strategy for functional regeneration and stem cell therapy. OBJECTIVE: To explore effects of transfected adult bone marrow clonogenic stem cells (AMCSCs) on cell phenotype, proliferation and cardiac differentiation potential. METHODS: Plasmid-encoded reporter gene maxGFP was used for nucleofection of AMCSCs with U-23 program. Growth curves of AMCSCs before and after nucleofection were compared based on results of MTT assay. AMCSCs before and after nucleofection were treated with 3 μmol/L 5-azacytidine for inducing cardiac differentiation. The cardiac differentiation specific markers, GATA4 and MLC-2v, were applied to confirm cardiac differentiation by RT-PCR. The maxGFP transfected AMCSCs were conducted the intramyocardium injection into an adult Sprague-Dawley rat left anterior descending coronary artery (LAD) ligation model to trace the in vivo expression of transfected maxGFP gene. Fluorescence images of the injected heart were analyzed on days 2 and 7 postinjection. RESULTS AND CONCLUSION: At 24 hours following nucleofection, the transfection efficiencies of AMCSCs at passages 47 and 119 were 49.4% and 43.1%. On 5 hours, green fluorescence positive cells were observed. Following nucleofection, AMCSCs maintained round shape, and could adhere and show clone-shape growth. MTT assay results demonstrated that the passages 47 and 119 of AMCSCs exhibited similar growth curves before and after nucleofection. Mean population doubling time was 8.57 and 10.28 hours in passages 47 and 119 of AMCSCs prior to nucleofection, and 9.42 and 10.42 hours following nucleofection (P =0.551, P=0.774). RT-PCR results showed that AMCSCs expressed GATA4 before and after 5-azacitidine treatment prior to nucleofection, and strongly expressed MLC-2v strip after treatment. AMCSCs expressed GATA4 prior to and following 5-azacitidine treatment after nucleofection, and strongly expressed MLC-2v after treatment. No significant difference was determined in above-mentioned indexes prior to and following nucleofection. In vivo experiment results demonstrated that a few green fluorescence positive cells were apparent in injected myocardium on days 2 and 7 following transfected AMCSCs injection. Results indicated that nucleofection is an effective and fast method for transfection of exogenous DNA into cell. The AMCSCs which are experienced with nucleofection are able to maintain their morphology, proliferation and cardiac differentiation potential. However, only a few transfected AMCSCs express the transferred gene, GFP, after intramyocardium injection.
Su X.,Peking Union Medical College |
Wu Z.,Peking Union Medical College |
Wu Z.,Huazhong University of Science and Technology |
Chen J.,Peking Union Medical College |
And 11 more authors.
Journal of Orthopaedic Research | Year: 2015
Cartilage-derived mesenchymal stem cells (MSCs) have been isolated with different methods. In this study lateral and medial femoral condyles were respectively collected from patients with late-stage osteoarthritis during the total knee arthroplasty. After digestion of the cartilage tissues with type II collagenase and analysis by fluorescence-activated cell sorting (FACS) with CD146, a chondroprogenitor cell sub-population were isolated and purified. The expression of other MSC-associated markers in the CD146 +chondroprogenitors was analyzed by flow cytometry. Multi-lineage differentiation capacity of CD146 + chondroprogenitors was compared with that of unsorted chondrocytes and adipose-derived MSCs (ADMSCs). Higher percentage of CD146 + chondroprogenitors isolated from the medial femoral condyles was observed than that from the lateral. CD146 + chondroprogenitors expressed high levels of MSC-specific surface antigens, and showed higher chondrogenesis capacity than ADMSCs and unsorted chondrocytes in a 3D cell pellet culture model. Thus CD146 might be a new cell surface marker for cartilage progenitor cell population in the late-stage osteoarthritis. © 2014 Orthopaedic Research Society.
Chen H.,Key Laboratory for Regenerative Medicine |
Chen H.,Chinese University of Hong Kong |
Peng P.,Key Laboratory for Regenerative Medicine |
Peng P.,Jinan University |
And 7 more authors.
International Journal of Cardiology | Year: 2012
Background: Coronary heart diseases (CHD) remain the most prevalent cause of premature death. Substantial growth of new collateral coronary vessels to the ischemic region would provide reconstitution of the occluded arteries and correction of heart ischemia. However, this remains an impossible mission with current advances. Methods: Incomplete ligation of left anterior descending (LAD) coronary artery was applied in rat resulting in partial occlusion of LAD. This chronic CHD model was employed to assess the therapeutic angiogenesis of Angio-T using ECG and echocardiography. Histological analysis was performed to provide substantial evidence for therapeutic angiogenesis in ischemic hearts with the possible involvement of JAK-STAT signaling pathway investigated. Results: Angio-T stimulated growth of new collateral microvessels in ischemic hearts and progressively improved heart functional performance 2 weeks post treatment. The involvement of JAK/STAT signaling pathway in Angio-T stimulated growth of new collateral coronary vessels in ischemic hearts was demonstrated. Conclusions: The substantial therapeutic angiogenesis of Angio-T in ischemic hearts was demonstrated that may provide a more effective solution for non-interventional treatment of chronic CHD. © 2010 Elsevier Ireland Ltd.
Nadeem D.,University of Bristol |
Smith C.-A.,University of Glasgow |
Dalby M.J.,University of Glasgow |
Dominic Meek R.M.,Southern General Hospital |
And 4 more authors.
Biofabrication | Year: 2015
Surface topography is known to influence stem cells and has been widely used as physical stimuli to modulate cellular behaviour including adhesion, proliferation and differentiation on 2D surfaces. Integration of well-defined surface topography into three-dimensional (3D) scaffolds for tissue engineering would be useful to direct the cell fate for intended applications. Technical challenges are remaining as how to fabricate such 3D scaffolds with controlled surface topography from a range of biodegradable and biocompatible materials. In this paper, a novel fabrication process using computer numerically controlled machining and lamination is reported to make 3D calcium phosphate/gelatin composite scaffolds with integrated surface micropatterns that are introduced by embossing prior to machining. Geometric analysis shows that this method is versatile and can be used to make a wide range of lattices with porosities that meet the basic requirements for bone tissue engineering. Both in vitro and in vivo studies show that micropatterned composite scaffolds with surfaces comprising 40 μm pits and 50 μm grooves were optimal for improved osteogenesis. The results have demonstrated the potential of a novel fabrication process for producing cell-instructive scaffolds with designed surface topographies to induce specific tissue regeneration. © 2015 IOP Publishing Ltd.
Yau W.W.Y.,Chinese University of Hong Kong |
Tang M.K.,Chinese University of Hong Kong |
Chen E.,Chinese University of Hong Kong |
YaoYao,Chinese University of Hong Kong |
And 4 more authors.
Proteome Science | Year: 2011
Background: Hair bulge progenitor cells (HBPCs) are multipotent stem cells derived from the bulge region of mice vibrissal hairs. The purified HBPCs express CD34, K15 and K14 surface markers. It has been reported that HBPCs could be readily induced to transdifferentiate into adipocytes and osteocytes. However, the ability of HBPCs to transdifferentiate into cardiomyocytes has not yet been investigated.Methodology/Principal Findings: The cardiomyogenic potential of HBPCs was investigated using a small cell-permeable molecule called Cardiogenol C. We established that Cardiogenol C could induce HBPCs to express transcription factors GATA4, Nkx2.5 and Tbx5, which are early specific markers for pre-cardiomyogenic cells. In prolonged cultures, the Cardiogenol C-treated HBPCs can also express muscle proteins, cardiac-specific troponin I and sarcomeric myosin heavy chain. However, we did not observe the ability of these cells to functionally contract. Hence, we called these cells cardiomyocyte-like cells rather than cardiomyocytes. We tried to remedy this deficiency by pre-treating HBPCs with Valproic acid first before exposing them to Cardiogenol C. This pretreatment inhibited, rather than improved, the effectiveness of Cardiogenol C in reprogramming the HBPCs. We used comparative proteomics to determine how Cardiogenol C worked by identifying proteins that were differentially expressed. We identified proteins that were involved in promoting cell differentiation, cardiomyocyte development and for the normal function of striated muscles. From those differentially expressed proteins, we further propose that Cardiogenol C might exert its effect by activating the Wnt signaling pathway through the suppression of Kremen1. In addition, by up-regulating the expression of chromatin remodeling proteins, SIK1 and Smarce1 would initiate cardiac differentiation.Conclusions/Significance: In conclusion, our CD34+/K15+HBPCs could be induced to transdifferentiate into cardiomyocyte-like cells using a small molecule called Cardiogenol C. The process involves activation of the Wnt signaling pathway and altered expression of several key chromatin remodeling proteins. The finding is clinically significant as HBPCs offer a readily accessible and autologous source of progenitor cells for cell-based therapy of heart disease, which is one of major killers in developed countries. © 2011 Yau et al; licensee BioMed Central Ltd.
Chen B.-F.,Key Laboratory for Regenerative Medicine |
Gu S.,Key Laboratory for Regenerative Medicine |
Suen Y.-K.,Key Laboratory for Regenerative Medicine |
Li L.,Key Laboratory for Regenerative Medicine |
Chan W.-Y.,Key Laboratory for Regenerative Medicine
Epigenetics | Year: 2014
It was previously demonstrated that miR-199a was downregulated in testicular germ cell tumor (TGCT), probably due to hypermethylation of its promoter. Further study found that re-expression of miR-199a in testicular cancer cells (NT2) led to suppression of cell growth, cancer migration, invasion and metastasis. More detailed analyses showed that these properties of miR-199a could be assigned to miR-199a-5p, one of its two derivatives. The biological role of the other derivative, miR-199a-3p in TGCT, remains largely uncharacterized. In this report, we identified DNA (cytosine-5)-methyltransferase 3A (DNMT3A), the de novo methyltransferase, as a direct target of miR-199a-3p using a 3'-UTR reporter assay. Transient expression of miR-199a-3p in NT2 cells led to decrease, while knocking down of miR-199a-3p in a normal human testicular cell line (HT) led to elevation, of DNMT3A2 (DNMT3A gene isoform 2) mRNA and protein levels. In clinical samples, DNMT3A2 was significantly overexpressed in malignant testicular tumor, and the expression of DNMT3A2 was inversely correlated with the expression of miR-199a-3p. However, DNMT3A did not affect miR-199a expression in NT2 cells. Further characterization of miR-199a-3p revealed that it negatively regulated DNA methylation, partly through targeting DNMT3A. Overexpression of miR-199a-3p restored the expression of APC and MGMT tumor-suppressor genes in NT2 cells by affecting DNA methylation of their promoter regions. Our studies demonstrated the deregulation of miR-199a-3p expression in TGCT may provide novel mechanistic insights into TGCT carcinogenesis and suggested a potentially therapeutic use of synthetic miR-199a-3p oligonucleotides as effective hypomethylating compounds in the treatment of TGCT. © 2014 Landes Bioscience.
Wang G.,Jinan University |
Wang G.,Quanzhou Medical College |
Yan Y.,Jinan University |
Chen X.,Jinan University |
And 3 more authors.
BioMed Research International | Year: 2014
The BCR-ABL kinase inhibitor imatinib is highly effective in the treatment of chronic myeloid leukemia (CML). However, long-term imatinib treatment induces immunosuppression, which is mainly due to T cell dysfunction. Imatinib can reduce TCR-triggered T cell activation by inhibiting the phosphorylation of tyrosine kinases such as Lck, ZAP70, LAT, and PLCγ1 early in the TCR signaling pathway. The purpose of this study was to investigate whether the superantigen SEA, a potent T cell stimulator, can block the immunosuppressive effects of imatinib on T cells. Our data show that the exposure of primary human T cells and Jurkat cells to SEA for 24 h leads to the upregulation of the Lck and ZAP70 proteins in a dose-dependent manner. T cells treated with SEA prior to TCR binding had increased the tyrosine phosphorylation of Lck, ZAP70, and PLCγ1. Pretreatment with SEA prevents the inhibitory effects of imatinib on TCR signaling, which leads to T cell proliferation and IL-2 production. It is conceivable that SEA antagonizes the imatinib-mediated inhibition of T cell activation and proliferation through the TCR signaling pathway. © 2014 Guanming Wang et al.