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Title: In vitro expansion of Lin{sup +} and Lin{sup −} mononuclear cells from human peripheral blood

Abstract

Haematopoietic stem cells (HSCs) are used in the therapy of blood disorders due to the ability of these cells to reconstitute haematopoietic lineage cells when transplanted into myeloablative recipients. However, substantial number of cells is required in order for the reconstitution to take place. Since HSCs present in low frequency, larger number of donor is required to accommodate the demand of transplantable HSCs. Therefore, in vitro expansion of HSCs will have profound impact on clinical purposes. The aim of this study was to expand lineage negative (Lin{sup −}) stem cells from human peripheral blood. Total peripheral blood mononuclear cells (PBMNCs) were fractionated from human blood by density gradient centrifugation. Subsequently, PBMNCs were subjected to magnetic assisted cell sorter (MACS) which depletes lineage positive (Lin{sup +}) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin{sup −} cell population. The ability of Lin{sup +} and Lin{sup −} to survive in vitro was explored by culturing both cell populations in complete medium consisting of Alpha-Minimal Essential Medium (AMEM) +10% (v/v) Newborn Calf Serum (NBCS)+ 2% (v/v) pen/strep. In another experiment, Lin{sup +} and Lin{sup −} were cultured with complete mediummore » supplemented with 10ng/mL of the following growth factors: stem cell factor (SCF), interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), 2IU/mL of Erythropoietin (Epo) and 20ng/mL of IL-6. Three samples were monitored in static culture for 22 days. The expansion potential was assessed by the number of total viable cells, counted by trypan blue exclusion assay. It was found that Lin{sup +} mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin{sup −} stem cells were not able to survive in proliferation medium however, addition of cytokines into the proliferation medium support Lin{sup −} stem cells for at least 18 days. The Lin{sup −} stem cells started to response to the cytokines added as early as Day 2 of culture. It is concluded that Lin{sup −} stem cells can be expanded in vitro by culturing in proliferation medium supplemented with cytokines.« less

Authors:
; ;  [1];  [2]
  1. School of Bioscience and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Selangor (Malaysia)
  2. Department of Orthodontics, Faculty of Dentistry, Universiti Kebangsaan Malaysia, 50300, Kuala Lumpur (Malaysia)
Publication Date:
OSTI Identifier:
22262698
Resource Type:
Journal Article
Resource Relation:
Journal Name: AIP Conference Proceedings; Journal Volume: 1571; Journal Issue: 1; Conference: 2013 UKM FST postgraduate colloquium, Selangor (Malaysia), 3-4 Jul 2013; Other Information: (c) 2013 AIP Publishing LLC; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ERYTHROPOIETIN; HUMAN POPULATIONS; IN VITRO; LEUKOCYTES; LYMPHOKINES; STEM CELLS; TRYPAN BLUE

Citation Formats

Norhaiza, H. Siti, Zarina, Z. A. Intan, Hisham, Z. A. Shahrul, and Rohaya, M. A. W. In vitro expansion of Lin{sup +} and Lin{sup −} mononuclear cells from human peripheral blood. United States: N. p., 2013. Web. doi:10.1063/1.4858661.
Norhaiza, H. Siti, Zarina, Z. A. Intan, Hisham, Z. A. Shahrul, & Rohaya, M. A. W. In vitro expansion of Lin{sup +} and Lin{sup −} mononuclear cells from human peripheral blood. United States. doi:10.1063/1.4858661.
Norhaiza, H. Siti, Zarina, Z. A. Intan, Hisham, Z. A. Shahrul, and Rohaya, M. A. W. Wed . "In vitro expansion of Lin{sup +} and Lin{sup −} mononuclear cells from human peripheral blood". United States. doi:10.1063/1.4858661.
@article{osti_22262698,
title = {In vitro expansion of Lin{sup +} and Lin{sup −} mononuclear cells from human peripheral blood},
author = {Norhaiza, H. Siti and Zarina, Z. A. Intan and Hisham, Z. A. Shahrul and Rohaya, M. A. W.},
abstractNote = {Haematopoietic stem cells (HSCs) are used in the therapy of blood disorders due to the ability of these cells to reconstitute haematopoietic lineage cells when transplanted into myeloablative recipients. However, substantial number of cells is required in order for the reconstitution to take place. Since HSCs present in low frequency, larger number of donor is required to accommodate the demand of transplantable HSCs. Therefore, in vitro expansion of HSCs will have profound impact on clinical purposes. The aim of this study was to expand lineage negative (Lin{sup −}) stem cells from human peripheral blood. Total peripheral blood mononuclear cells (PBMNCs) were fractionated from human blood by density gradient centrifugation. Subsequently, PBMNCs were subjected to magnetic assisted cell sorter (MACS) which depletes lineage positive (Lin{sup +}) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin{sup −} cell population. The ability of Lin{sup +} and Lin{sup −} to survive in vitro was explored by culturing both cell populations in complete medium consisting of Alpha-Minimal Essential Medium (AMEM) +10% (v/v) Newborn Calf Serum (NBCS)+ 2% (v/v) pen/strep. In another experiment, Lin{sup +} and Lin{sup −} were cultured with complete medium supplemented with 10ng/mL of the following growth factors: stem cell factor (SCF), interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), 2IU/mL of Erythropoietin (Epo) and 20ng/mL of IL-6. Three samples were monitored in static culture for 22 days. The expansion potential was assessed by the number of total viable cells, counted by trypan blue exclusion assay. It was found that Lin{sup +} mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin{sup −} stem cells were not able to survive in proliferation medium however, addition of cytokines into the proliferation medium support Lin{sup −} stem cells for at least 18 days. The Lin{sup −} stem cells started to response to the cytokines added as early as Day 2 of culture. It is concluded that Lin{sup −} stem cells can be expanded in vitro by culturing in proliferation medium supplemented with cytokines.},
doi = {10.1063/1.4858661},
journal = {AIP Conference Proceedings},
number = 1,
volume = 1571,
place = {United States},
year = {Wed Nov 27 00:00:00 EST 2013},
month = {Wed Nov 27 00:00:00 EST 2013}
}
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