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, 8 (9), e74361
eCollection

Busulfan Administration Flexibility Increases the Applicability of Scid Repopulating Cell Assay in NSG Mouse Model

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Busulfan Administration Flexibility Increases the Applicability of Scid Repopulating Cell Assay in NSG Mouse Model

Jean Chevaleyre et al. PLoS One.

Abstract

Background: Xenotransplantation models allowing the identification and quantification of human Hematopoietic stem cells (HSC) in immunodeficient mice remain the only way to appropriately address human HSC function despite the recent progress in phenotypic characterization. However, these in vivo experiments are technically demanding, time consuming and expensive. Indeed, HSCs engraftment in mouse requires pre-conditioning of animals either by irradiation or cytotoxic drugs to allow homing of injected cells in specific stem cell niches and their subsequent expansion and differentiation in bone marrow. Recently, the development of busulfan pre-conditioning of animals improved the flexibility of experimentation in comparison with irradiation.

Design and methods: In order to further facilitate the organization of these complex experiments we investigated the effect of extending the period between mice pre-conditioning and cell injection on the engraftment efficiency. In the meantime, we also explored the role of busulfan doses, mouse gender and intravenous injection route (caudal or retro orbital) on engraftment efficiency.

Results and conclusion: We showed that a period of up to 7 days did not modify engraftment efficiency of human HSCs in NSG model. Moreover, retro orbital cell injection to female mice pre-conditioned with 2x25 mg/kg of busulfan seems to be the best adapted schema to detect the human HSC in xenotransplantation experiments.

Conflict of interest statement

Competing Interests: The authors have read the journal’s policy and declare the following interest: co-author Dr Zoran IVANOVIC is a PLOS ONE Editorial Board Member. However, this does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Evaluation of busulfan pre-conditioning in mice.
Mice received increasing doses of busulfan (2 injections) and were weighted prior and after 24 hours following each injection. A. Percentage (%) of weight relative to weight prior injection (---) and measured 24 hours after the first (▪) and the second (□) injection of different doses of busulfan. B. Percentage of weight loss according to gender 24 hours after the first (▪) and the second (□) injection. C. Survival of mice according to gender. D-E. Mice received 1000 non manipulated CD34+ cells by i.v. caudal injection and were analyzed 8 weeks later for huCD45 chimerism (D) and huCFU content (E) in their bone marrow. Each mouse represented by (●), Median represented by (─).
Figure 2
Figure 2. Impact on engraftment level of time delay between mice pre-conditioning and cell injections.
Female mice received 2 doses of busulfan (25mg/Kg each) at 24 hours, 4 days or 7 days intervals. Mice received 1000 non manipulated CD34+ cells by caudal injection and their bone marrow were analyzed 8 weeks later for A. total huCD45 chimerism, B. B lymphoid (▪) versus myeloid (□) cells ration in CD45+ cells, and C. huCFUs content per femur. Each mouse represented by (●), Median represented by (─).
Figure 3
Figure 3. Comparison of caudal vein and retro orbital injections.
After classical busulfan pre-conditioning (2x25 mg/Kg), female mice received 1000 non manipulated CD34+ cells by i.v. injection either in the caudal vein or retro orbital sinus. Their bone marrow were analyzed 8 weeks later for A. total huCD45 chimerism, B. huCFU content. Each mouse represented by (●), Median represented by (─).
Figure 4
Figure 4. Impact of gender on human cells engraftment.
After classical busulfan pre-conditioning (2x25 mg/Kg), female and male mice received 100, 500 or 1000 non manipulated CD34+ cells by i.v. injection in the retro orbital sinus. Their bone marrow were analyzed 8 weeks later for A. total huCD45 chimerism, B. huCFU content. Each mouse represented by ● (female) or ○ (male), Median represented by (─).

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Grant support

The source of funding for this work is the French Blood Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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