scholarly journals Characterization of Transplanted Green Fluorescent Protein+Bone Marrow Cells into Adipose Tissue

Stem Cells ◽  
2008 ◽  
Vol 26 (2) ◽  
pp. 330-338 ◽  
Author(s):  
Koji Tomiyama ◽  
Noriko Murase ◽  
Donna Beer Stolz ◽  
Hideyoshi Toyokawa ◽  
Daniel R. O'Donnell ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Adipose Tissue ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Bone Marrow Cells ◽  
Green Fluorescent ◽  
Marrow Cells

scholarly journals Enhanced Green Fluorescent Protein as Selectable Marker of Retroviral-Mediated Gene Transfer in Immature Hematopoietic Bone Marrow Cells

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3304-3315 ◽  
Author(s):  
Marti F.A. Bierhuizen ◽  
Yvonne Westerman ◽  
Trudi P. Visser ◽  
Wati Dimjati ◽  
Albertus W. Wognum ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Fluorescent Protein ◽  
Bone Marrow Cells ◽  
Retroviral Vector ◽  
Egfp Expression ◽  
Green Fluorescent ◽  
Marrow Cells

Abstract The further improvement of gene transfer into hematopoietic stem cells and their direct progeny will be greatly facilitated by markers that allow rapid detection and efficient selection of successfully transduced cells. For this purpose, a retroviral vector was designed and tested encoding a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP). Murine cell lines (NIH 3T3, Rat2) and bone marrow cells transduced with this retroviral vector demonstrated a stable green fluorescence signal readily detectable by flow cytometry. Functional analysis of the retrovirally transduced bone marrow cells showed EGFP expression in in vitro clonogenic progenitors (GM-CFU), day 13 colony-forming unit-spleen (CFU-S), and in peripheral blood cells and marrow repopulating cells of transplanted mice. In conjunction with fluorescence-activated cell sorting (FACS) techniques EGFP expression could be used as a marker to select for greater than 95% pure populations of transduced cells and to phenotypically define the transduced cells using antibodies directed against specific cell-surface antigens. Detrimental effects of EGFP expression were not observed: fluorescence intensity appeared to be stable and hematopoietic cell growth was not impaired. The data show the feasibility of using EGFP as a convenient and rapid reporter to monitor retroviral-mediated gene transfer and expression in hematopoietic cells, to select for the genetically modified cells, and to track these cells and their progeny both in vitro and in vivo.

Identification of Leukemia Stem Cells Associated with AML1-ETO through Characterization of Oncogene-Induced Changes in Cell Surface Antigen Profiles on Hematopoietic Stem Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1357-1357
Author(s):  
Carmen S Ballestas ◽  
Hyung-Gyoon Kim ◽  
Claude Scott Swindle ◽  
Christopher Klug
Keyword(s):  
Bone Marrow ◽  
Green Fluorescent Protein ◽  
Cell Surface ◽  
Progenitor Cell ◽  
Fluorescent Protein ◽  
Bone Marrow Cells ◽  
Retroviral Vector ◽  
Cell Phenotype ◽  
Green Fluorescent ◽  
Marrow Cells

Abstract Acute myelogenous leukemia (AML) is a heterogenous group of myeloid malignancies that are characterized by the clonal outgrowth of immature myeloid progenitor cells. For most subtypes of AML, mutations that give rise to the leukemic phenotype occur in the hematopoietic stem/progenitor cell (HSC) subset as demonstrated by studies showing that only primitive CD34+CD38− bone marrow cells could function as leukemia-initiating cells (LSC) when transferred into immunodeficient NOD-SCID mice. One rather significant challenge has been that LSC share many of the same cell-surface markers as their normal counterparts in bone marrow, thus making it difficult to functionally characterize and purify this important subset of leukemic cells from bulk bone marrow samples. To rapidly identify novel antigens that are mutation-specific and induced specifically on LSC and not on normal HSC, we have transduced highly purified HSC isolated from mouse bone marrow with retroviral vectors that co-expressed AML1-ETO along with a green fluorescent protein (GFP) reporter gene. HSC of the cell-surface phenotype c-Kit+Lin−Sca-1+Flt3− (KLSF) were transduced for 18 hours in vitro and then were resorted for GFP+ cells by flow cytometry (FACS). Purified cells were then lysed for mRNA isolation and cDNA synthesis for the generation of a probe that was then hybridized to Affymetrix oligonucleotide arrays (430 2.0 GeneChip arrays). HSC transduced with a retroviral vector that only expressed GFP were used as controls to identify genes that would normally be expressed in the HSC subset. Importantly, since retroviral vectors only integrate into cycling cells, all sorted GFP+ cells from the independent transductions would represent cycling cells, which minimizes any gene expression differences due to differential frequencies of resting versus actively cycling HSC. Changes in expression of cell-surface proteins observed at the mRNA level were then validated at the protein level using FACS. Bone marrow cells were isolated from an animal that was transplanted with cells expressing AML1-ETO and GFP from a retroviral vector. Cells were stained for the HSC/progenitor cell phenotype (KLS) as well as for the cell-surface marker of interest. For one marker, CD55, we noted a 100-fold increase in cell-surface expression specifically on HSC that express AML1-ETO and not on normal HSC. These results indicate that short-term retroviral expression of specific AML-associated mutations in HSC followed by microarray analysis of transduced cells may provide a rapid means of prospectively identifying leukemia-initiating cells in bulk patient bone marrow samples and that CD55 may be a useful therapeutic and diagnostic marker for patient samples that express the AML1-ETO chromosomal translocation. Figure 1. CD55 expression distinguishes normal HSC from HSC with the AML1-ETO translocation. Bone marrow cells were isolated from an animal that was transplanted with cells expressing AML1-ETO and the green fluorescent protein (GFP) from a retroviral vector. Cells were stained for the HSC/progenitor cell phenotype (KLS) as well as for CD55. Cells that expressed AML1-ETO are shown below as GFP+ gated cells. Note that cells that express AML1-ETO express CD55 at approximately 100-fold greater levels on the cell-surface than GFP-negative (AML1-ETO-negative) bone marrow cells. MPC=myeloid progenitor cells of the phenotype c-Kit+ Lin− Sea-1− cells. HSC are defined c-Kit+ Lin− Sca-1+ cells. Figure 1. CD55 expression distinguishes normal HSC from HSC with the AML1-ETO translocation. Bone marrow cells were isolated from an animal that was transplanted with cells expressing AML1-ETO and the green fluorescent protein (GFP) from a retroviral vector. Cells were stained for the HSC/progenitor cell phenotype (KLS) as well as for CD55. Cells that expressed AML1-ETO are shown below as GFP+ gated cells. Note that cells that express AML1-ETO express CD55 at approximately 100-fold greater levels on the cell-surface than GFP-negative (AML1-ETO-negative) bone marrow cells. MPC=myeloid progenitor cells of the phenotype c-Kit+ Lin− Sea-1− cells. HSC are defined c-Kit+ Lin− Sca-1+ cells.

Presence of bone marrow–derived circulating progenitor endothelial cells in the newly formed lymphatic vessels

Blood ◽  
2005 ◽  
Vol 106 (13) ◽  
pp. 4184-4190 ◽  
Author(s):  
Piotr Religa ◽  
Renhai Cao ◽  
Meit Bjorndahl ◽  
Zhongjun Zhou ◽  
Zhenping Zhu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Fluorescent Protein ◽  
Bone Marrow Cells ◽  
Lymphatic Vessels ◽  
Growth Factor Receptor ◽  
Donor Bone Marrow ◽  
Green Fluorescent ◽  
Corneal Lymphangiogenesis ◽  
Marrow Cells

Bone marrow (BM)-derived circulating endothelial precursor cells (CEPCs) have been reported to incorporate into newly formed blood vessels under physiologic and pathologic conditions. However, it is unknown if CEPCs contribute to lymphangiogenesis. Here we show that in a corneal lymphangiogenesis model of irradiated mice reconstituted with enhanced green fluorescent protein (EGFP)-positive donor bone marrow cells, CEPCs are present in the newly formed lymphatic vessels. Depletion of bone marrow cells by irradiation remarkably suppressed lymphangiogenesis in corneas implanted with fibroblast growth factor-2 (FGF-2). Further, transplantation of isolated EGFP-positive/vascular endothelial growth factor receptor-3-positive (EGFP+/VEGFR-3+) or EGFP+/VEGFR-2+ cell populations resulted in incorporation of EGFP+ cells into the newly formed lymphatic vessels. EGFP+/CEPCs were also present in peritumoral lymphatic vessels of a fibrosarcoma. These data suggest that BM-derived CEPCs may play a role in “lymphvasculogenesis.”

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