Blood Res.  2016 Mar;51(1):31-36. 10.5045/br.2016.51.1.31.

Humanizing NOD/SCID/IL-2Rγnull (NSG) mice using busulfan and retro-orbital injection of umbilical cord blood-derived CD34+ cells

• Kang YK
• Ko Y
• Choi A
• Choi HJ
• Seo JH
• Lee M
• Lee JA

• KMID: 2172742
• DOI: http://doi.org/10.5045/br.2016.51.1.31

Abstract

BACKGROUND
Humanized mouse models are still under development, and various protocols exist to improve human cell engraftment and function.
METHODS
Fourteen NOD/SCID/IL-2Rγnull (NSG) mice (4"’5 wk old) were conditioned with busulfan and injected with human umbilical cord blood (hUCB)-derived CD34+ hematopoietic stem cells (HSC) via retro-orbital sinuses. The bone marrow (BM), spleen, and peripheral blood (PB) were analyzed 8 and 12 weeks after HSC transplantation.
RESULTS
Most of the NSG mice tolerated the regimen well. The percentage of hCD45+ and CD19+ cells rose significantly in a time-dependent manner. The median percentage of hCD45+cells in the BM was 55.5% at week 8, and 67.2% at week 12. The median percentage of hCD45+ cells in the spleen at weeks 8 and 12 was 42% and 51%, respectively. The median percentage of hCD19+ cells in BM at weeks 8 and 12 was 21.5% and 39%, respectively (P=0.04). Similarly, the median percentage of hCD19+ cells in the spleen at weeks 8 and 12 was 10% and 24%, respectively (P=0.04). The percentage of hCD19+ B cells in PB was 23% at week 12. At week 8, hCD3+ T cells were barely detectable, while hCD7+ was detected in the BM and spleen. The percentage of hCD3+ T cells was 2"’3% at week 12 in the BM, spleen, and PB of humanized NSG mice.
CONCLUSION
We adopted a simplified protocol for establishing humanized NSG mice. We observed a higher engraftment rate of human CD45+ cells than earlier studies without any significant toxicity. And human CD45+ cell engraftment at week 8 was comparable to that of week 12.

Keyword

Humanized mice; Busulfan; Retro-orbital sinus; Hematopoietic stem cell

MeSH Terms

Animals
B-Lymphocytes
Bone Marrow
Busulfan*
Fetal Blood
Hematopoietic Stem Cells
Humans*
Mice*
Spleen
T-Lymphocytes
Umbilical Cord*
Busulfan

Figure

• 

  Fig. 1 Scheme for generating the humanized NSG mice. MNCs isolated from hUCB were enriched using a RosetteSep kit and human CD34 MicroBead Kit. NSG mice were conditioned by busulfan and CD34+ cells were injected via retro-orbital sinus.

• 

  Fig. 2 Survival and weight changes of the NSG mice after hCD34+ cell injection. Humanized NSG mice were monitored daily after transplantation. Most of the NSG mice did well, but one mice (depicted with an arrow) showed features suggesting GVHD (weight loss, hunched posture and diminished activity as shown in the photo) 10 days after transplantation.

• 

  Fig. 3 Human cell reconstitution of NSG mice transplanted with hUCB-derived CD34+ cells. Levels of human CD45+ cells in mouse tissues at different times after transplantation are shown. Bone marrow, spleen, and blood were isolated from the humanized NSG mice and MNCs isolated from each organ were stained and analyzed. The percentages are represented as mean±SEM in humanized mice.

• 

  Fig. 4 Human CD19+ and CD3+ cell reconstitution from NSG mice injected with hUCB-derived CD34+ cells. The percentages are represented by mean±SEM in humanized mice.

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  Fig. 5 hCD45 and hCD7 expression levels in spleen (SPL) and bone marrow (BM) of NSG mice 8 weeks after hUCB-derived CD34+ cell injection.

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