Transfer protocol of human HSC into NOG mice
Mice:
Adult NOG mice are aged 8 - 12 weeks. Newborn mice are 1 – 2 days old.
Irradiation:
Irradiation is performed with 2 - 2.5 Gy to adult mice and with 1 Gy tonewborns one day prior to cell transfer under SPF conditions. Mice less than 10g body weight may die with this dose irradiation.

8-12 week old NOG mice
irradiated with 2.5 Gy

Human cell reconstituted
NOG
Intravenous transfer of1 - 0.5 x 105 hCD34+ cells

Newborn NOG mice
irradiated with 1 Gy

Intrahepatic or intravenous transferof 1 - 0.1 x 105 hCD34+ cells

8 - 20 weeks after celltransfer

Cell preparation
1. When CD34+ cells (e.g. 1 x 106/tube) are frozen, cells are thawed in a37℃ water bath.
2. Transfer cells into 50 mL conical tube and add 1 mL of PBS containing
2% fetal bovine serum (2%FBS-PBS) drop by drop shaking the tubeslowly.
3. After further addition of 18 mL of 2%FBS-PBS, centrifuge it at 1,200rpm for 5 min at room temperature.
4. Re-suspend the cells in 10 mL of 2%FBS-PBS, re-centrifuge.
5. Re-suspend in 2 mL of PBS.
6. Using 15 µL of the suspension, count the cell number and viability after
adding 15 µL of 0.25% trypan-blue solution. Add a further 3 mL of
PBS to reach a total of 5 mL.
7. Transfer 1 mL of the cell suspension into each 1.5 mL cryotube.
8. Put the tubes into a 50 mL conical tube and carry it to the animalfacility.
9. After dipping it in an antiseptic solution, carry it into the animal room.
Cell transfer
For adult mice
1. 0.25 mL (1 - 0.5 x 104) of the cell suspension is injected into mice via
the tail vein with a 1 mL syringe with 27 G or a Microinjector syringewith 29 G under slight anesthesia with Isoflurane.
For newborn mice
1. Slightly anesthetize the newborn with Isoflurane.
2. Hold the newborn with left hand so the head of the mouse face right.
3. 0.1 mL (1-5 x 104 ) of the cell suspension is injected via the face veinwith a Microinjector syringe with 29 G.
4. Return the newborn to the mother gently, do not give it a shock.

Engraftment of human cell in NOG mice

8 - 20 weeks after intravenous transplantation of 5 x 104 human cord blood derived CD34+ cells into NOG and NOD-scid mice, humanCD45+ hematopoietic cells in peripheral blood of mice were detected in flow cytometry. The high engraftment rate of human cells wasobserved in NOG mice.

In order to evaluate the frequency of CB CD34+ cells capable of engrafting NOG mice, these mice were transplanted with CB CD34+
cells in a limiting dose. All recipient mice transplanted with 1 x 103 CB CD34+ cells showed successful engraftment (>0.1%). Further, as
few as 100 cells could be engrafted in 2 of 6 mice and their multi-lineage differentiation.

Multi-lineage cell differentiation from HSCs in NOG mice
In bone marrow
The rate of human CD45+ cells in bone marrow of transplanted mice andthe high efficacy of multi-lineage cell differentiation. 11 weeks after

CD34+ cell transplantation, bone marrow were removed fromNOD/SCID/γcnull and NOD/Shi-scid mice treated with anti-asialo
GM1 antibody two days before transplantation, and subjected toflow cytometry. NOD/SCID/γcnull showed significantly higher
percentage of CD45+ cells. Multi-lineage cells have beendifferentiated from CD34+ cells with high efficacy (* vs **:P<0.01).

1. Ito, M., H. Hiramatsu, K. Kobayashi, K. Suzue, M. Kawahata, K. Hioki, Y.
Ueyama, Y. Koyanagi, K. Sugamura, K. Tsuji, T. Heike, and T. Nakahata.
2002. NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse modelfor engraftment of human cells. Blood 100:3175-3182.
2. Yahata, T., K. Ando, Y. Nakamura, Y. Ueyama, K. Shimamura, N. Tamaoki,
S. Kato, and T. Hotta. 2002. Functional human T lymphocyte developmentfrom cord blood CD34+ cells in nonobese diabetic/Shi-scid, IL-2 receptor
gamma null mice. J Immunol 169:204-209.
3. Saito, Y., Y. Kametani, K. Hozumi, N. Mochida, K. Ando, M. Ito, T. Nomura,Y. Tokuda, H. Makuuchi, T. Tajima, and S. Habu. 2002. The in vivo
development of human T cells from CD34(+) cells in the murine thymic
environment. Int Immunol 14:1113-1124.
4. Hiramatsu, H., R. Nishikomori, T. Heike, M. Ito, K. Kobayashi, K. Katamura,and T. Nakahata. 2003. Complete reconstitution of human lymphocytes fromcord blood CD34+ cells using the NOD/SCID/gammacnull mice model. Blood
102:873-880.
5. Watanabe, Y., T. Takahashi, A. Okajima, M. Shiokawa, N. Ishii, I. Katano, R.
Ito, M. Ito, M. Minegishi, N. Minegishi, S. Tsuchiya, and K. Sugamura. 2009.
The analysis of the functions of human B and T cells in humanized NOD/shiscid/{gamma}cnull (NOG) mice (hu-HSC NOG mice). Int Immunol.
from Ito, M. et al. NOD/SCID/gamma(c)(null) mouse: an excellent recipientmouse model for engraftment of human cells. Blood 100, 3175-3182. (2002).

Development of lymphoid lineage cells in NOG micetransferred with human CD34+ cells

LeukocytesA

2 months

mouse CD45

blood
69.1%

B
spleen

blood
8.4%

15.5%
18.8%

4 months

Cspleen

13.1%
26.1%

43.9%

B cell characters

7 months
blood

10.3%
52.3%

67.7%

spleen

74.8%

6.7%

53.8%

81.9%

CD5IgD
IgM

human CD45

CD19

T and B cells
human CD19

blood

spleen

17.6%

41.7%
0.9%0.7%

bloodspleen

15.0%

22.2%

3.3%

blood
3.6%

5.1%

T cell charactersspleen
31.5%

14.9%

29.3%

human CD3
In the development of human cells from HSCs in NOG mice, B cells develop 4-8weeks after transfer. T cells can be detected in 8-12 weeks after transfer and later
become dominant in the periphery including peripheral blood and spleen.

from Watanabe, S. et al. Hematopoietic stem cell-engrafted NOD/SCID/IL2Rgammanull mice develop human lymphoid systems and induce long-lasting HIV-1 infection
with specific humoral immune responses. Blood 109, 212-218 (2007).

CD4αβ TCR
CD8

The above figure shows human cells in mouse
peripheral blood 20 weeks after intravenous human HSC
cell transfer. Human T and B cells were developed inNOG mice. IgM+IgD+ B cells and CD4+ and CD8+ Tcells were also differentiated in NOG mice.
from Dr. Habu S, Tokai Univ.

Development of myeloid lineage cells in NOG micetransferred with human CD34+ cells

Monocytehuman CD14

bloodspleen

0.6%

BM
1.1%

61.5%

9.0%

63.6%

72.3%

Megakaryocyte

PlateletIn bone marrow

In peripheral bloodH HHM

Humanplatelet
1.5%

M

HM
human CD45

H M

M

HhCD41M

hCD42b staining
H; human megakaryocytes
M; mouse megakaryocytes

Macrophage
LungLymph nodefrom Dr. Miyakawa Y, Keio Univ.

Mast cell
Non-irradiated
NOG

Irradiated NOG

In spleen
CD68

CD68

from Watanabe, S. et al. Hematopoietic stem cell-engrafted
NOD/SCID/IL2Rgamma null mice develop human lymphoid systems andinduce long-lasting HIV-1 infection with specific humoral immune responses.
Blood 109, 212-218 (2007).

(x10)

(x40)
hTryptase staining

Development of lymphoid follicle in NOG mice reconstituted
human CD34+ cells

SpleenHLA staining

human CD45

CD20 B cells

FDC-M1 murine FDC

CD3 T cells

CD68 macrophages

CD205 DC

Human lymphoid follicle like structures (White arrow) were observed in spleen ofNOG mice 18 wks after human HSC transfer.

from Watanabe, S. et al. Hematopoietic stem cell-engrafted NOD/SCID/IL2Rgamma null micedevelop human lymphoid systems and induce long-lasting HIV-1 infection with specifichumoral immune responses. Blood 109, 212-218 (2007).

Human B, T cells and other monocytes/macrophages and DCs, but not
FDCs were associated with the generation of lymphoid follicle-likestructures observed in spleen.

Analysis of T cell receptor Vβ repertoire

TCR Vß repertoire analysis of human T cells in spleen.
Experiment 1%

Experiment 2Experiment 3

20

15

10

50

12

3

4

5.1

5.2

5.3 7.1

7.2

891112 13.1 13.2 13.6 1416171820 21.3 22Vβ
Four to six months after transplantation of 2×104 to 5×104 CB CD34+ cells, spleen cells were taken and theTCR Vß repertoire was analyzed by flow cytometry using a panel of 24 different antibodies. The results ofthree independent experiments are shown.
from Hiramatsu, H., R. Nishikomori, T. Heike, M. Ito, K. Kobayashi, K. Katamura, and T. Nakahata. 2003.
Complete reconstitution of human lymphocytes from cord blood CD34+ cells using the NOD/SCID/gammacnull
mice model. Blood 102:873-880.

23Activation of human T cells developed in NOG mice
Proliferation and cytokine responses

Activation markers

hCD4 T cellfrom hPBMC

23.9CD40LcountcounthCD4 T cell fromNOG spleen68.854.8

30.0

42.0

CD25

59.9

CD69

from Dr. Habu S, Tokai Univ.

Antibody production in NOG mice transferred human HSCs

Immunization
DNP-KLH/alum via I.P.

day0NOG

10w

booster

12w

14w

16w

ELISA

Irradiation andHSC transplantation

Non-specific antibodies

Weeks

Anti-DNP-KLH specific antibodies

NOG/hCD34+

BALB/cAfrom Dr. Habu S, Tokai Univ.

Summary of human hematopoietic cells differentiatedfrom CB CD34+ cells in NOG mice

Erythrocytes ±Megakaryocytes (platellet) +MEP(Megakaryocyte/erythroidprogenitor)

Granulocytes (Neutrophils/Eosinophils/Basophils) ±Mast cells +Monocytes +CMP (Commonmyeloid progenitor)
HSC

GMP (Granulocyte/monocyteprogenitor)

Macrophages +DCs +B lymphocytes ++T lymphocytes ++CLP (Commonlymphoid progenitor)
Progenitor cells

NK cells +Mature blood cells