Murine stem cell gene marker analysis of longterm reconstitution and self-renewal

V. Feng, Vijaya Ayengar, Micheal Collector, Sara Neutzel, Linzhao Cheng, Saul J. Sharkis

Research output: Contribution to journalArticle

Abstract

Using either whole bone marrow or activated stem cells (i.e. 5FU treated) gene marking for engraftment and self- renewal in the mouse has been demonstrated. In this study we isolated a population containing an enriched stem cell population (FR25Lin) as an alternative approach to stem cell gene marking. FR25Lin cells were isolated from B6D2F, mouse bone marrow by elutriation and flow cytometry. This stem cell population was stimulated by 50ng/ml TPO, SCF, Flt-3 Ligand and 50U/ml IL-6 and transfected with the retroviral vector MGIN containing enhanced green fluorescent protein (EGFP). Then the cells were spinoculated with viral supernatant and continually incubated with cytokines. The precentage of EGFP+ cultured FR25Lin cells was 54%, 38%, 23%, and 30% at Day 2, 6, 11,16 alter viral infection. Progenitor cells grown out of these cultures were 53±4%, 53±7%, 39±6% and 66±10% positive for EGFP at these same time points. Six days after viral infection, (3.25× 106cells/mouse) the cultured cells were transplanted into lethally irradiated recipients. Five months later, the bone marrow cells were pooled from the recipients and further fractionated by elutriation. Whole marrow was 23% EGFP+ and the FR25 (approximately 25% of the whole marrow) was about the same percent positive (21%). Whole bone marrow, or EGFP+ selected cells from these primary recipients' FR25 population (collected by FACS) were injected into secondary recipients. Two and half months later the peripheral blood samples of the secondary recipients were analyzed for EGFP fluorescence. All four mice receiving 106 whole bone marrow cells showed EGFP+ RBC and WBC, ranging from 9% to 25% and from 24% to 41% respectively. Six of 7 recipients receiving 105 FR25 EGFP+ cells/mouse showed EGFP+ marked WBC (2% to 27%) and 3 of these recipients had EGFP marked RBC ( 1 % to 11 %). At a dose of l O4 FR25 EGFP+ cells/mouse, the peripheral blood samples of only 4 out of 8 recipients were positive for EGFP in only the WBC ( 1 % to 14%). We speculate that we can infect stem cells that can give rise to both myeloid and erythroid cells and that self-renewal of these stem cells can result in integration of the gene in multilineage as well as restricted stem/progenitor cells in secondary recipients. We conclude that EGFP as a gene marker can identify progeny of a stem cell enriched population that has undergone self-renewal and differentiation.

Original languageEnglish (US)
JournalBlood
Volume96
Issue number11 PART II
StatePublished - 2000

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Stem cells
Stem Cells
Genes
Bone
Bone Marrow
Cells
Population
Virus Diseases
Bone Marrow Cells
enhanced green fluorescent protein
Cultured Cells
Blood
Erythroid Cells
Flow cytometry
Myeloid Cells
Cell culture
Fluorouracil
Interleukin-6
Flow Cytometry
Fluorescence

ASJC Scopus subject areas

  • Hematology

Cite this

Feng, V., Ayengar, V., Collector, M., Neutzel, S., Cheng, L., & Sharkis, S. J. (2000). Murine stem cell gene marker analysis of longterm reconstitution and self-renewal. Blood, 96(11 PART II).

Murine stem cell gene marker analysis of longterm reconstitution and self-renewal. / Feng, V.; Ayengar, Vijaya; Collector, Micheal; Neutzel, Sara; Cheng, Linzhao; Sharkis, Saul J.

In: Blood, Vol. 96, No. 11 PART II, 2000.

Research output: Contribution to journalArticle

Feng, V, Ayengar, V, Collector, M, Neutzel, S, Cheng, L & Sharkis, SJ 2000, 'Murine stem cell gene marker analysis of longterm reconstitution and self-renewal', Blood, vol. 96, no. 11 PART II.
Feng V, Ayengar V, Collector M, Neutzel S, Cheng L, Sharkis SJ. Murine stem cell gene marker analysis of longterm reconstitution and self-renewal. Blood. 2000;96(11 PART II).
Feng, V. ; Ayengar, Vijaya ; Collector, Micheal ; Neutzel, Sara ; Cheng, Linzhao ; Sharkis, Saul J. / Murine stem cell gene marker analysis of longterm reconstitution and self-renewal. In: Blood. 2000 ; Vol. 96, No. 11 PART II.
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abstract = "Using either whole bone marrow or activated stem cells (i.e. 5FU treated) gene marking for engraftment and self- renewal in the mouse has been demonstrated. In this study we isolated a population containing an enriched stem cell population (FR25Lin) as an alternative approach to stem cell gene marking. FR25Lin cells were isolated from B6D2F, mouse bone marrow by elutriation and flow cytometry. This stem cell population was stimulated by 50ng/ml TPO, SCF, Flt-3 Ligand and 50U/ml IL-6 and transfected with the retroviral vector MGIN containing enhanced green fluorescent protein (EGFP). Then the cells were spinoculated with viral supernatant and continually incubated with cytokines. The precentage of EGFP+ cultured FR25Lin cells was 54{\%}, 38{\%}, 23{\%}, and 30{\%} at Day 2, 6, 11,16 alter viral infection. Progenitor cells grown out of these cultures were 53±4{\%}, 53±7{\%}, 39±6{\%} and 66±10{\%} positive for EGFP at these same time points. Six days after viral infection, (3.25× 106cells/mouse) the cultured cells were transplanted into lethally irradiated recipients. Five months later, the bone marrow cells were pooled from the recipients and further fractionated by elutriation. Whole marrow was 23{\%} EGFP+ and the FR25 (approximately 25{\%} of the whole marrow) was about the same percent positive (21{\%}). Whole bone marrow, or EGFP+ selected cells from these primary recipients' FR25 population (collected by FACS) were injected into secondary recipients. Two and half months later the peripheral blood samples of the secondary recipients were analyzed for EGFP fluorescence. All four mice receiving 106 whole bone marrow cells showed EGFP+ RBC and WBC, ranging from 9{\%} to 25{\%} and from 24{\%} to 41{\%} respectively. Six of 7 recipients receiving 105 FR25 EGFP+ cells/mouse showed EGFP+ marked WBC (2{\%} to 27{\%}) and 3 of these recipients had EGFP marked RBC ( 1 {\%} to 11 {\%}). At a dose of l O4 FR25 EGFP+ cells/mouse, the peripheral blood samples of only 4 out of 8 recipients were positive for EGFP in only the WBC ( 1 {\%} to 14{\%}). We speculate that we can infect stem cells that can give rise to both myeloid and erythroid cells and that self-renewal of these stem cells can result in integration of the gene in multilineage as well as restricted stem/progenitor cells in secondary recipients. We conclude that EGFP as a gene marker can identify progeny of a stem cell enriched population that has undergone self-renewal and differentiation.",
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