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An improved protocol for efficient engraftment in NOD/LTSZ-SCIDIL-2Rγ mice allows HIV replication and development of anti-HIV immune responses.

Singh M, Singh P, Gaudray G, Musumeci L, Thielen C, Vaira D, Vandergeeten C, Delacroix L, Van Gulck E, Vanham G, de Leval L, Rahmouni S, Moutschen M - PLoS ONE (2012)

Bottom Line: In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3-4 week old NSG mice.Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days.We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection.

View Article: PubMed Central - PubMed

Affiliation: Immunology and infectious diseases unit GIGA-I3, University of Liege, Liege, Belgium.

ABSTRACT
Cord blood hematopoietic progenitor cells (CB-HPCs) transplanted immunodeficient NOD/LtsZ-scidIL2Rγ() (NSG) and NOD/SCID/IL2Rγ() (NOG) mice need efficient human cell engraftment for long-term HIV-1 replication studies. Total body irradiation (TBI) is a classical myeloablation regimen used to improve engraftment levels of human cells in these humanized mice. Some recent reports suggest the use of busulfan as a myeloablation regimen to transplant HPCs in neonatal and adult NSG mice. In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3-4 week old NSG mice. In this CB-CD34+transplanted NSG mice engraftment efficiency of human CD45+cell is over 90% in peripheral blood. Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days. These humanized NSG mice have shown long-lasting viremia after HIV-1JRCSF and HIV-1Bal inoculation through intravenous and rectal routes. We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection. Humanized NSG mice reconstituted according to our new protocol produced, moderate cellular and humoral immune responses to HIV-1 postinfection. We believe that NSG mice reconstituted according to our easy to use protocol will provide a better in vivo model for HIV-1 replication and anti-HIV-1 therapy trials.

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Engraftment potential of CD34+ hematopoietic cells.(A) Flow cytometric analysis of murine and human CD45+ cells in the peripheral blood of NSG mice of two different groups, representative profiles of the mice engraftment levels after 12 and 22 weeks after CD34+ cells transplantation. (B) Engraftment levels of human CD45+ cells in peripheral blood up to 306 days after transplantation in group 1 and group 7. (C) White blood cell counts at 12 and 22 weeks post-engraftment in group 7 mice along with human control.
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pone-0038491-g001: Engraftment potential of CD34+ hematopoietic cells.(A) Flow cytometric analysis of murine and human CD45+ cells in the peripheral blood of NSG mice of two different groups, representative profiles of the mice engraftment levels after 12 and 22 weeks after CD34+ cells transplantation. (B) Engraftment levels of human CD45+ cells in peripheral blood up to 306 days after transplantation in group 1 and group 7. (C) White blood cell counts at 12 and 22 weeks post-engraftment in group 7 mice along with human control.

Mentions: Previous protocols for humanization of NSG or NOG mice required TBI before transplantation of CB-HPCs in order to obtain sufficient CD45+ human cell engraftment [5], [6], [7]. Alternative protocols using busulfan as a myelosuppressive agent in neonate NSG mice have also been described [12], [13], [14], [15]. Although busulfan indeed improves human CD45+ cell engraftment levels in humanized NSG mice, some limitation persists in the published protocols (i.e. need to transfer large numbers of CB–CD34+ cells, cost of cytokine treatment, difficulty of i.v. injections in mice neonates and limited availability of human fetal tissue) [12], [14], [15]. With the goal to improve busulfan-based humanization protocols, we first treated a group of twenty 8–9 week old mice (group 1) with i.p. injection of busulfan 20 mg/kg, 24 hours prior to i.v.transplantation with 1×105 frozen CB-CD34+ cells. We then evaluated engraftment levels by iterative analysis of the blood of engrafted mice. We did not observe any mortality with this dose of busulfan. Engraftment levels of human CD45+ cell at week 22 were 41.26±5.70% (n = 20) and stable engraftment persisted up to 300 days (Fig. 1A and 1B).


An improved protocol for efficient engraftment in NOD/LTSZ-SCIDIL-2Rγ mice allows HIV replication and development of anti-HIV immune responses.

Singh M, Singh P, Gaudray G, Musumeci L, Thielen C, Vaira D, Vandergeeten C, Delacroix L, Van Gulck E, Vanham G, de Leval L, Rahmouni S, Moutschen M - PLoS ONE (2012)

Engraftment potential of CD34+ hematopoietic cells.(A) Flow cytometric analysis of murine and human CD45+ cells in the peripheral blood of NSG mice of two different groups, representative profiles of the mice engraftment levels after 12 and 22 weeks after CD34+ cells transplantation. (B) Engraftment levels of human CD45+ cells in peripheral blood up to 306 days after transplantation in group 1 and group 7. (C) White blood cell counts at 12 and 22 weeks post-engraftment in group 7 mice along with human control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366932&req=5

pone-0038491-g001: Engraftment potential of CD34+ hematopoietic cells.(A) Flow cytometric analysis of murine and human CD45+ cells in the peripheral blood of NSG mice of two different groups, representative profiles of the mice engraftment levels after 12 and 22 weeks after CD34+ cells transplantation. (B) Engraftment levels of human CD45+ cells in peripheral blood up to 306 days after transplantation in group 1 and group 7. (C) White blood cell counts at 12 and 22 weeks post-engraftment in group 7 mice along with human control.
Mentions: Previous protocols for humanization of NSG or NOG mice required TBI before transplantation of CB-HPCs in order to obtain sufficient CD45+ human cell engraftment [5], [6], [7]. Alternative protocols using busulfan as a myelosuppressive agent in neonate NSG mice have also been described [12], [13], [14], [15]. Although busulfan indeed improves human CD45+ cell engraftment levels in humanized NSG mice, some limitation persists in the published protocols (i.e. need to transfer large numbers of CB–CD34+ cells, cost of cytokine treatment, difficulty of i.v. injections in mice neonates and limited availability of human fetal tissue) [12], [14], [15]. With the goal to improve busulfan-based humanization protocols, we first treated a group of twenty 8–9 week old mice (group 1) with i.p. injection of busulfan 20 mg/kg, 24 hours prior to i.v.transplantation with 1×105 frozen CB-CD34+ cells. We then evaluated engraftment levels by iterative analysis of the blood of engrafted mice. We did not observe any mortality with this dose of busulfan. Engraftment levels of human CD45+ cell at week 22 were 41.26±5.70% (n = 20) and stable engraftment persisted up to 300 days (Fig. 1A and 1B).

Bottom Line: In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3-4 week old NSG mice.Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days.We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection.

View Article: PubMed Central - PubMed

Affiliation: Immunology and infectious diseases unit GIGA-I3, University of Liege, Liege, Belgium.

ABSTRACT
Cord blood hematopoietic progenitor cells (CB-HPCs) transplanted immunodeficient NOD/LtsZ-scidIL2Rγ() (NSG) and NOD/SCID/IL2Rγ() (NOG) mice need efficient human cell engraftment for long-term HIV-1 replication studies. Total body irradiation (TBI) is a classical myeloablation regimen used to improve engraftment levels of human cells in these humanized mice. Some recent reports suggest the use of busulfan as a myeloablation regimen to transplant HPCs in neonatal and adult NSG mice. In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3-4 week old NSG mice. In this CB-CD34+transplanted NSG mice engraftment efficiency of human CD45+cell is over 90% in peripheral blood. Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days. These humanized NSG mice have shown long-lasting viremia after HIV-1JRCSF and HIV-1Bal inoculation through intravenous and rectal routes. We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection. Humanized NSG mice reconstituted according to our new protocol produced, moderate cellular and humoral immune responses to HIV-1 postinfection. We believe that NSG mice reconstituted according to our easy to use protocol will provide a better in vivo model for HIV-1 replication and anti-HIV-1 therapy trials.

Show MeSH
Related in: MedlinePlus