Đại học Quốc gia Thành phố Hồ Chí Minh

Trường Đại học Khoa học tự nhiên

PTN Nghiên cứu và Ứng dụng Tế bào gốc

Category: Bài báo ngoài nước

  • Hypoxia promotes adipose-derived stem cell proliferation via VEGF

    Hypoxia promotes adipose-derived stem cell proliferation via VEGF

    Phuc Van Pham, Ngoc Bich Vu, Ngoc Kim Phan

    Abstract

    Adipose-derived stem cells (ADSCs) are a promising mesenchymal stem cell source with therapeutic applications. Recent studies have shown that ADSCs could be expanded in vitro without phenotype changes. This study aimed to evaluate the effect of hypoxia on ADSC proliferation in vitro and to determine the role of vascular endothelial growth factor (VEGF) in ADSC proliferation. ADSCs were selectively cultured from the stromal vascular fraction obtained from adipose tissue in DMEM/F12 medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic. ADSCs were cultured under two conditions: hypoxia (5% O2) and normal oxygen (21% O2). The effects of the oxygen concentration on cell proliferation were examined by cell cycle and doubling time. The expression of VEGF was evaluated by the ELISA assay. The role of VEGF in ADSC proliferation was studied by neutralizing VEGF with anti-VEGF monoclonal antibodies. We found that the ADSC proliferation rate was significantly higher under hypoxia compared with normoxia. In hypoxia, ADSCs also triggered VEGF expression. However, neutralizing VEGF with anti-VEGF monoclonal antibodies significantly reduced the proliferation rate. These results suggest that hypoxia stimulated ADSC proliferation in association with VEGF production.

    Keywords

    Adipose derived stem cells; Hypoxia; Mesenchymal stem cell; VEGF

    Full Text:

    PDF

  • Culture and differentiation of cytokine-induced killer cells from umbilical cord blood-derived monon

    Culture and differentiation of cytokine-induced killer cells from umbilical cord blood-derived mononuclear cells

    Binh Thanh Vu, Quyen Thanh-Ngoc Duong, Phong Minh Le, Phuc Van Pham

    Abstract

    Cytokine-induced killer cells (CIK) are cytotoxic T cells, which have both NK and T cell properties. These cells are characterized by potent, non-MHC-restricted cytotoxicity and reduced alloreactivity, which make them appealing for use in adoptive immunotherapy of cancer and virus infections. In this study, CIK cells were generated by stimulating umbilical cord blood-derived mononuclear cells (UCB-MNCs) with interferon-gamma (IFN-g) on day 0. Anti-CD3 antibody and interleukin-2 (IL-2) were added after 24 hours at four different experimental concentration combinations in order to identify the optimal cytokine amounts for CIK cell proliferation. Cells were collected at four time points over a 21-day period (day 0, 7, 14, 21) for analysis of cell marker presentation using flow cytometry, as well as transcription-level cytokine production using RT-PCR. The results showed that in the 21-day culture, the average final expansion levels of CD3+CD56+ CIK cell were in the range of hundredfold, accounted for 26% in the bulk culture. Most important, these cells strongly expressed granzyme B (80.87%), a potent factor involved in cell-mediated cytotoxicity. These CIK cells also transcriptionally overexpressed the three cytokine genes that produce IFN-g, tumor necrosis factor-alpha (TNF-a), and IL-2; these are key for immune cell mobilization against tumors as well as foreign pathogens. Our research establishes an effective cytokine concentration and time protocol for use in generation of CIK cells from UCB-MNCs, potentiating greater applications of CIK cell-adoptive immunotherapy in both research and clinical settings. Thus, the 3­rd and 4th experimental conditions both stimulated CIK cell differentiation with 50 ng/ml of anti-CD3 antibody, but with IL-2 concentrations of 500 U/ml and 1000 U/ml, respectively.

    Keywords

    Cytokine-induced killer, immunotherapy, non-MHC restricted, umbilical cord blood-MNC.

    Full Text:

    PDF

  • Preliminary evaluation of treatment efficacy of umbilical cord blood-derived mesenchymal stem cell-d

    Preliminary evaluation of treatment efficacy of umbilical cord blood-derived mesenchymal stem cell-differentiated cardiac progenitor cells in a myocardial injury mouse model

    Truc Le-Buu Pham, Tam Thanh Nguyen, Anh Thi-Van Bui, Ho Thanh Pham, Ngoc Kim Phan, My Thi-Thu Nguyen, Phuc Van Pham

    Abstract

    Recently, stem cell therapy has been investigated as a strategy to prevent or reverse damage to heart tissue. Although the results of cell transplantation in animal models and patients with myocardial ischemia are promising, the selection of the appropriate cell type remains an issue that requires consideration. In this study, we aimed to evaluate the effect of cardiac progenitor cell transplantation in a mouse model of myocardial ischemia. The cardiac progenitor cells used for transplantation were differentiated from umbilical cord blood mesenchymal stem cells. Animal models injected with phosphate-buffered saline (PBS) and healthy mice were used as controls. Cell grafting was assessed by changes in blood pressure and histological evaluation. After 14 days of transplantation, the results demonstrated that the blood pressure of transplanted mice was stable, similar to healthy mice, whereas it fluctuated in PBS-injected mice. Histological analysis showed that heart tissue had regenerated in transplanted mice, but remained damaged in PBS-injected mice. Furthermore, trichrome staining revealed that the transplanted mice did not generate significant amount of scar tissue compared with PBS-injected control mice. In addition, the cardiac progenitor cells managed to survive and integrate with local cells in cell-injected heart tissue 14 days after transplantation. Most importantly, the transplanted cells did not exhibit tumorigenesis. In conclusion, cardiac progenitor cell transplantation produced a positive effect in a mouse model of myocardial ischemia.

  • Low concentrations of 5-aza-2′-deoxycytidine induce breast cancer stem cell differentiation by trigg

    Low concentrations of 5-aza-2′-deoxycytidine induce breast cancer stem cell differentiation by triggering tumor suppressor gene expression

    Nhan Lu-Chinh Phan, Ngu Van Trinh, Phuc Van Pham

    Laboratory of Stem Cell Research and Application, University of Science, Vietnam National University, Ho Chi Minh City, Vietnam

    Background: Breast cancer stem cells (BCSCs) are considered the cause of tumor growth, multidrug resistance, metastasis, and recurrence. Therefore, differentiation therapy to reduce self-renewal of BCSCs is a promising approach. We have examined the effects of 5-aza-2′-deoxycytidine (DAC) on BCSC differentiation.
    Materials and methods: BCSCs were treated with a range of DAC concentrations from 0.625 to 100 µM. The differentiation status of DAC-treated BCSCs was graded by changes in cell proliferation, CD44+CD24- phenotype, expression of tumor suppressor genes, including BRCA1, BRCA2, p15, p16, p53, and PTEN, and antitumor drug resistance.
    Results: DAC treatment caused significant BCSC differentiation. BCSCs showed a 15%–23% reduction in proliferation capacity, 3.0%–21.3% decrease in the expression of BCSC marker CD44+/CD24-, activation of p53 expression, and increased p15, p16, BRCA1, and BRCA2 expression. Concentrations of DAC ranging from 0.625 to 40 µM efficiently induce cell cycle arrest in S-phase. ABCG2, highly expressed in BCSCs, also decreased with DAC exposure. Of particular note, drug-sensitivity of BCSCs to doxorubicin, verapamil, and tamoxifen also increased 1.5-, 2.0-, and 3.7-fold, respectively, after pretreatment with DAC.
    Conclusion: DAC reduced breast cancer cell survival and induced differentiation through reexpression of tumor suppressor genes. These results indicate the potential of DAC in targeting specific chemotherapy-resistant cells within a tumor.

    Keywords: breast cancer, breast cancer stem cells, differentiation, epigenetics, 5-aza-2′-deoxycytidine

     

    Download Article [PDF]

     

  • Isolation and proliferation of umbilical cord tissue derived mesenchymal stem cells for clinical app

    Isolation and proliferation of umbilical cord tissue derived mesenchymal stem cells for clinical applications

    • Phuc Van Pham Email author
    • , Nhat Chau Truong
    • , Phuong Thi-Bich Le
    • , Tung Dang-Xuan Tran
    • , Ngoc Bich Vu
    • , Khanh Hong-Thien Bui
    • , Ngoc Kim Phan

    Abstract

    Umbilical cord (UC) is a rich source of rapidly proliferating mesenchymal stem cells (MSCs) that are easily cultured on a large-scale. Clinical applications of UC–MSCs include graft-versus-host disease, and diabetes mellitus types 1 and 2. UC–MSCs should be isolated and proliferated according to good manufacturing practice (GMP) with animal component-free medium, quality assurance, and quality control for their use in clinical applications. This study developed a GMP standard protocol for UC-MSC isolation and culture. UC blood and UC were collected from the same donors. Blood vasculature was removed from UC. UC blood was used as a source of activated platelet rich plasma (aPRP). Small fragments (1–2 mm2) of UC membrane and Wharton’s jelly were cut and cultured in DMEM/F12 medium containing 1 % antibiotic–antimycotic, aPRP (2.5, 5, 7.5 and 10 %) at 37 °C in 5 % CO2. The MSC properties of UC–MSCs at passage 5 such as osteoblast, chondroblast and adipocyte differentiation, and markers including CD13, CD14, CD29, CD34, CD44, CD45, CD73, CD90, CD105, and HLA-DR were confirmed. UC–MSCs also were analyzed for karyotype, expression of tumorigenesis related genes, cell cycle, doubling time as well as in vivo tumor formation in NOD/SCID mice. Control cells consisted of UC–MSCs cultured in DMEM/F12 plus 1 % antibiotic–antimycotic, and 10 % fetal bovine serum (FBS). All UC-MSC (n = 30) samples were successfully cultured in medium containing 7.5 and 10 % aPRP, 92 % of samples grew in 5.0 % aPRP, 86 % of samples in 2.5 % aPRP, and 72 % grew in 10 % FBS. UC–MSCs in these four groups exhibited similar marker profiles. Moreover, the proliferation rates in medium with PRP, especially 7.5 and 10 %, were significantly quicker compared with 2.5 and 5 % aPRP or 10 % FBS. These cells maintained a normal karyotype for 15 sub-cultures, and differentiated into osteoblasts, chondroblasts, and adipocytes. The analysis of pluripotent cell markers showed UC–MSCs maintained the expression of the oncogenes Nanog and Oct4 after long term culture but failed to transfer tumors in NOD/SCID mice. Replacing FBS with aPRP in the culture medium for UC tissues allowed the successful isolation of UC–MSCs that satisfy the minimum standards for clinical applications.

    Keywords

    Activated platelet rich plasma Clinical application of mesenchymal stem cells Umbilical cord Umbilical cord derived mesenchymal stem cells Good manufacturing practice UC–MSCs

    Title
    Isolation and proliferation of umbilical cord tissue derived mesenchymal stem cells for clinical applications
    Journal
    Cell and Tissue Banking

    DOI
    10.1007/s10561-015-9541-6
    Print ISSN
    1389-9333
    Online ISSN
    1573-6814
    Publisher
    Springer Netherlands
    Additional Links
    Topics
    Keywords
    • Activated platelet rich plasma
    • Clinical application of mesenchymal stem cells
    • Umbilical cord
    • Umbilical cord derived mesenchymal stem cells
    • Good manufacturing practice
    • UC–MSCs
    Industry Sectors
    Authors
    Author Affiliations
    • 1. Laboratory of Stem Cell Research and Application, University of Science, Vietnam National University, Ho Chi Minh City, Vietnam
    • 2. Van Hanh Stem Cell Unit, Van Hanh Hospital, Ho Chi Minh City, Vietnam
    • 3. University Medical Center, University of Medicine and Pharmacy, Ho Chi Minh City, Vietnam
  • Production of dendritic cells and cytokine-induced killer cells from banked umbilical cord blood sam

    Production of dendritic cells and cytokine-induced killer cells from banked umbilical cord blood sam

    Production of dendritic cells and cytokine-induced killer cells from banked umbilical cord blood samples

    Abstract

    Umbilical cord blood (UCB) is considered to be a source of hematopoietic stem cells (HSCs). All UCB banks have recently become interested in the isolation and storage of HSCs for the treatment of hematological diseases. However, UCB was also recently confirmed as a source of immune cells for immunotherapy such as dendritic cells (DCs) and cytokine-induced killer cells (CIKs). This study aimed to exploit this source of immune cells in banked UCB samples. After collection of UCB samples, mononuclear cells (MNCs) containing stem cells, progenitor cells, and mature cells were isolated by Ficoll-Hypaque-based centrifugation. The MNCs were subjected to freezing and thawing according to a previously published protocol. The banked MNCs were used to produce DCs and CIKs. To produce DCs, MNCs were induced in RPMI 1640 medium supplemented with GM-CSF (50 ng/ml) and IL-4 (40 ng/ml) for 14 days. To produce CIKs, MNCs were induced in RPMI 1640 medium supplemented an anti-CD3 monoclonal antibody, IL-3, and GMC-SF for 21–28 days. Both DCs and CIKs were evaluated for their phenotypes and functions according to previously published protocols. The results showed that banked UCB samples can be successfully used to produce functional DCs and CIKs. These samples are valuable sources of immune cells for immunotherapy. The present results suggest that banked UCB samples are useful not only for stem cell isolation, but also for immune cell production.

    Keywords

    Banked UCB Stem cells Immune cells Dendritic cells Cytokine-induced killer cells Immunotherapy

    Title
    Production of dendritic cells and cytokine-induced killer cells from banked umbilical cord blood samples
    Open Access
    Available under Open Access
    Journal
    Biomedical Research and Therapy
    2:28

    Online Date
    November 2015
    DOI
    10.7603/s40730-015-0028-7
    Online ISSN
    2198-4093
    Publisher
    Laboratory of Stem Cell Research and Application
    Additional Links
    Topics
    Keywords
    • Banked UCB
    • Stem cells
    • Immune cells
    • Dendritic cells
    • Cytokine-induced killer cells
    • Immunotherapy
    Authors
    Author Affiliations
    • 1. Laboratory of Stem Cell Research and Application, University of Science, Viet Nam National University, Ho Chi Minh, Viet Nam

  • Comparison of the Treatment Efficiency of Bone Marrow-Derived Mesenchymal Stem Cell Transplantation

    Stem Cells International
    Article ID 5720413

    Comparison of the Treatment Efficiency of Bone Marrow-Derived Mesenchymal Stem Cell Transplantation via Tail and Portal Veins in CCl4-Induced Mouse Liver Fibrosis

    Nhung Hai Truong,1,2 Nam Hai Nguyen,1 Trinh Van Le,1 Ngoc Bich Vu,1 Nghia Huynh,3 Thanh Van Nguyen,4 Huy Minh Le,3 Ngoc Kim Phan,1,2 and Phuc Van Pham1,2

    1Laboratory of Stem cell Research and Application, University of Science, VNU-HCM, Ho Chi Minh City 700000, Vietnam
    2Biology Faculty, University of Science, VNU-HCM, Ho Chi Minh City 700000, Vietnam
    3University of Medicine and Pharmacy Ho Chi Minh City, Ho Chi Minh City 700000, Vietnam
    4Nguyen Tat Thanh University, Ho Chi Minh City, Vietnam

    Received 3 June 2015; Revised 15 September 2015; Accepted 17 September 2015

    Academic Editor: Shinn-Zong Lin

    Copyright © 2016 Nhung Hai Truong et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

     


    Abstract

    Because of self-renewal, strong proliferation in vitro, abundant sources for isolation, and a high differentiation capacity, mesenchymal stem cells are suggested to be potentially therapeutic for liver fibrosis/cirrhosis. In this study, we evaluated the treatment effects of mouse bone marrow-derived mesenchymal stem cells (BM-MSCs) on mouse liver cirrhosis induced by carbon tetrachloride. Portal and tail vein transplantations were examined to evaluate the effects of different injection routes on the liver cirrhosis model at 21 days after transplantation. BM-MSCs transplantation reduced aspartate aminotransferase/alanine aminotransferase levels at 21 days after injection. Furthermore, BM-MSCs induced positive changes in serum bilirubin and albumin and downregulated expression of integrins (600- to 7000-fold), transforming growth factor, and procollagen-α1 compared with the control group. Interestingly, both injection routes ameliorated inflammation and liver cirrhosis scores. All mice in treatment groups had reduced inflammation scores and no cirrhosis. In conclusion, transplantation of BM-MSCs via tail or portal veins ameliorates liver cirrhosis in mice. Notably, there were no differences in treatment effects between tail and portal vein administrations. In consideration of safety, we suggest transfusion of bone marrow-derived mesenchymal stem cells via a peripheral vein as a potential method for liver fibrosis treatment.

    Nhung Hai Truong, Nam Hai Nguyen, Trinh Van Le, et al., “Comparison of the Treatment Efficiency of Bone Marrow-Derived Mesenchymal Stem Cell Transplantation via Tail and Portal Veins in CCl4-Induced Mouse Liver Fibrosis,” Stem Cells International, Article ID 5720413, in press.

    http://www.hindawi.com/journals/sci/aa/5720413/cta/

     

  • Vitamin C stimulates human gingival stem cell proliferation and expression of pluripotent markers

    In Vitro Cellular & Developmental Biology – Animal

    pp 1-10

    First online: 20 October 2015

    Vitamin C stimulates human gingival stem cell proliferation and expression of pluripotent markers

    • Phuc Van Pham
    • , Nga Yen Tran
    • , Nhan Lu-Chinh Phan
    • , Ngoc Bich Vu
    • , Ngoc Kim Phan

     

    Abstract

    Gingival stem cells (GSCs) are a novel source of mesenchymal stem cells (MSCs) that are easily accessed from the oral cavity. GSCs were considered valuable autograft MSCs with particular characteristics. However, the limitation in the number of available GSCs remains an obstacle. Therefore, this study aimed to stimulate GSC proliferation by ascorbic acid (AA) and determined the effects of AA on GSC pluripotent potential-related gene expression. GSCs were isolated from gum tissue by explant culture and continuously subcultured before analysis of stemness and effects of AA on pluripotent-related gene expression. GSCs cultured with various concentrations of AA showed increased proliferation in a dose-dependent manner. AA-treated GSCs showed significantly higher expression of SSEA-3, Sox-2, Oct-3/4, Nanog, and TRA-1-60 compared with control cells. More importantly, GSCs also maintained their stemness with MSC phenotypes and failed to cause tumors in nude athymic mice. Our results show that AA is a suitable factor to stimulate GSC proliferation.

    Keywords

    Gingival stem cell Gum stem cell Mesenchymal stem cell Pluripotent marker

    Title
    Vitamin C stimulates human gingival stem cell proliferation and expression of pluripotent markers
    Journal
    In Vitro Cellular & Developmental Biology – Animal

    DOI
    10.1007/s11626-015-9963-2
    Print ISSN
    1071-2690
    Online ISSN
    1543-706X
    Publisher
    Springer US
    Additional Links
    Topics
    Keywords
    • Gingival stem cell
    • Gum stem cell
    • Mesenchymal stem cell
    • Pluripotent marker
    Industry Sectors
    Authors
    Author Affiliations
    • 1. Laboratory of Stem Cell Research and Application, University of Science, Viet Nam National University, Ho Chi Minh City, Viet Nam
    • 2. Faculty of Dentistry, University of Medicine and Pharmacy, Ho Chi Minh City, Viet Nam
    http://link.springer.com/article/10.1007/s11626-015-9963-2

  • ISOLATION AND CHARACTERIZATION OF MULTIPOTENT AND PLURIPOTENT STEM CELLS FROM HUMAN PERIPHERAL BLOOD

    ISOLATION AND CHARACTERIZATION OF MULTIPOTENT AND PLURIPOTENT STEM CELLS FROM HUMAN PERIPHERAL BLOOD

    Article · July 2015

    Ciro Gargiulo
    Human Medicine International Center

    Phuc Van Pham
    Ho Chi Minh City University of Science

    Abstract

    Stem cells are commonly classified based on the developmental stage from which they are isolated, although this has been a source of debate amongst stem cell scientists. A common approach classifies stem cells into three different groupings: Embryonic Stem Cells (ESCs), Umbilical Cord Stem Cells (UCBSCs) and Adult Stem Cells (ASCs), which includes stem cells from bone marrow (BM), fat tissue (FT), engineered induced pluripotent (IP) and peripheral blood (PB). By definition stem cells are progenitor cells capable of self-renewal and differentiation hypothetically “ab infinitum” into more specialized cells and tissue. The main intent of this study was to determine and characterize the different sub-groups of stem cells present within the human PB-SCs that may represent a valid opportunity in the field of clinical regenerative medicine. Stem cells in the isolated mononucleated cells were characterized using a multidisciplinary approach that was based on morphology, the expression of stem cell markers by flowcytometry and fluorescence analysis, RT-PCR and the capacity to self-renew or proliferate and differentiate into specialized cells. This approach was used to identify the expression of hematopoietic, mesenchymal, embryonic and neural stem cell markers. Both isolated adherent and suspension mononucleated cells were able to maintain their stem cell properties during in-vitro culture by holding their capacity for proliferation and differentiation into osteoblast cells, respectively, when exposed to the appropriate induction medium.

  • A comparison of the chemical and liver extract-induced hepatic differentiation of adipose derived st

    A comparison of the chemical and liver extract-induced hepatic differentiation of adipose derived stem cells

    Article in In Vitro Cellular & Developmental Biology – Animal · August 2015
    Impact Factor: 1.00 · DOI: 10.1007/s11626-015-9939-2

    Truong Nhung
    Ho Chi Minh City University of Science

    Phuc Van Pham
    Ho Chi Minh City University of Science

    Abstract

    Adipose-derived stem cells (ADSCs) have been put forward as promising therapeutics for end-stage liver disease (ESLD). In the present study, we compared the effects of defined chemicals and liver extract on the hepatic differentiation of ADSCs. ADSCs were isolated according to the method described in our previously published study. Subsequently, the differentiation of ADSCs was induced separately by chemicals (including hepatic growth factor (HGF), fibroblast growth factor (FGF), and oncostatin M (OSM)) and liver extract (30 μg/ml) in a total period of 21 d. The efficiency of hepatic differentiation was evaluated by changes in the cell morphology, gene expression, and cellular function. The results showed that the liver extract promoted the hepatic differentiation of ADSCs to a significantly greater extent than the chemicals. In the group of ADSCs treated with liver extract, changes in the cell morphology began sooner, and the expression of alpha-FP and albumin genes was higher than that in the chemically treated group. The ADSCs in both the groups stained positive for anti-alpha trypsin (AAT) and albumin markers. The cells also exhibited glycogen storage capacity. Therefore, we concluded that the liver extract could efficiently induce the differentiation of ADSCs into hepatocyte-like cells. This study reveals the potential of mesenchymal stem cell differentiation in the liver extract, which supports further preclinical and clinical research on the application of ADSCs in ESLD treatment.