Other models• Model with human ovary
Terada, Y., Y. Terunuma-Sato, T. Kakoi-Yoshimoto, H. Hasegawa, T. Ugajin, Y. Koyanagi, M. Ito, T.
Murakami, H. Sasano, N. Yaegashi, and K. Okamura. 2008. Development of human Graafian follicles
following transplantation of human ovarian tissue into NOD/SCID/gammac null mice. Am J Reprod
Immunol 60:534-540.
Transplantation of human ovarian cortex into host mice may permit various kinds of challenges in reproductive
medicine. A novel immunodeficient mouse strain (NOD/SCID/gammac null: NOG) has been developed as a hostfor transplantation of human tissue. Human ovarian cortex was transplanted into various sites of NOG mice andhuman follicular development was examined by immunohistochemistry. Transplantation of human ovarian tissue
into NOG mice resulted in approximately similar tissue survival and follicle growth as did transplantation into
non-obese diabetic-severe combined immunodeficient mice. The human Graafian follicle from NOG mouse
expressed the same steroidogenic enzymes as observed in human Graafian follicles, which developed in thehuman body. The NOG mice's ovarian bursa was better placed for transplantation than the back skin or kidney
capsule. These results represent the successful generation and biological confirmation of the human Graafianfollicles from the human ovarian cortex in the NOG mice.
Figure 1. Macroscopic view of a human
Graafian follicle (arrow) after
transplantation of human ovarian
cortex into the ovarian bursa of a NOG
mouse.

kidneyHost mice were stimulated by daily
intraperitoneal injection of human menopausal
gonadotropin for 14 days, 10 weeks after
transplantation. Scale bar = 1 cm.

Human ovary

Figure 2. Characterization of a human
Graafian follicle that developed in theovarian bursa of a NOG mouse.

G TGT(b)

(a)

GT
G(c)

T(d)

(a) Hematoxylin and eosin staining showing thetheca and granulosa cell layers. (b) Antibody
staining for the steroidogenic enzyme P450 scc
is localized to the cytoplasm of theca cells. (c)Immunohistochemistry for AD4-BP showing
expression in the nuclei of both theca andgranulosa cell layers. (d) ER antibody staining
was detected in the theca cell layer. T, thecacells; G, granulosa cells. Scale bar = 100 µm.

Other models• Model with human endometrium
Matsuura-Sawada, R., T. Murakami, Y. Ozawa, H. Nabeshima, J. Akahira, Y. Sato, Y. Koyanagi, M. Ito, Y.
Terada, and K. Okamura. 2005. Reproduction of menstrual changes in transplanted human endometrial tissue inimmunodeficient mice. Hum Reprod 20:1477-1484.
Cultures of human endometrial tissue are useful for analysing the mechanisms underlying the menstrual cycle. However,long-term culture of endometrial tissue is difficult in vitro. Xenotransplantation of normal human endometrial tissue into
immunodeficient mice could allow prolonged survival of the transplanted tissues. Proliferative-phase endometrial tissue
samples from three women were transplanted into the subcutaneous space of ovariectomized, immunodeficient, non-obese
diabetic (NOD)/severe combined immunodeficiency (SCID)/gammaC(null) (NOG) mice. The mice were treated with
17beta-estradiol (E2) for the first 14 days after transplantation, followed by E2 plus progesterone for the next 14 days.
The transplants were investigated morphologically and immunohistochemically at various times after implantation. The
transplanted tissues contained large numbers of small glands, pseudostratification of the nuclei and dense stroma aftertreatment with E2 alone. After treatment with E2 plus progesterone, subnuclear vacuolation, luminal secretion anddecidualization of the stroma were observed. When the hormone treatment ceased, tissue destruction occurred and thetransplants returned to the proliferative phase. Lymphocytes were identified immunohistochemically: the numbers of
CD56-positive and CD16-negative cells increased significantly in the stroma during the late secretory phase (day 28).
Human endometrial tissue transplanted into NOG mice showed similar histological changes to eutopic endometrial tissueduring treatment with sex steroid hormones for 1 month. Moreover, lymphocytes were produced in the transplanted
human endometrial tissue. This system represents a new experimental model of the human endometrium in vivo.

Figure 1. Histological sections stained with haematoxylin andeosin (A–G) and the immunohistochemical stain Ki-67 (H).
(A) Pretransplantation endometrial tissue: the proliferative phase. Theglands are small straight and narrow, with columnar glandular cells andprominent pseudostratification of the nuclei. The stromal cells are dense(magnification ×400). (B) Endometrial tissue 14 days after transplantation:
E2 has been administered for 14 days. The glands are small and narrow,with tall columnar cells. Evidence of pseudostratification of the nuclei ispresent. The stromal cells are dense (magnification ×400). (C) Endometrial
tissue 16 days after transplantation: E2 has been administered for 14 days,followed by E2 plus progesterone for 2 days. The glands still show
pseudostratified structures but they have begun to enlarge, and subnuclear
vacuolation of the glandular cells is visible. The stromal cells remain dense(magnification ×400). (D) Endometrial tissue 21 days after transplantation:
E2 has been administered for 14 days, followed by E2 plus progesterone for7 days. The glands are significantly dilated, the glandular cells are cuboidal
and the pseudostratification of the nuclei has disappeared. Stromal
decidualization is beginning. Many lymphocytes are present throughout thestroma and are aggregating around the glands (magnification ×200). (E)Endometrial tissue 28 days after transplantation: E2 has been administeredfor 14 days, followed by E2 plus progesterone for 14 days. The glandularcells are cuboidal. Evidence of stromal decidualization is clearly seen andlymphocytes are present in the stroma (magnification ×200). (F)Endometrial tissue 31 days after transplantation: E2 had been administeredfor 14 days, followed by E2 plus progesterone for 14 days, and then nohormones for the remaining 3 days. The glands and endometrial stroma
have collapsed during the evolution of the transplant. There is prominent
bleeding in the stroma (magnification ×200). (G) Endometrial tissue 35
days after transplantation: E2 had been administered for 14 days, followedby E2 plus progesterone for 14 days, and then no hormones for theremaining 7 days. The glands are small and narrow with tall columnar cells.
Evidence of pseudostratification of the nuclei is present. The stromal cells
are dense (magnification ×400). (H) Endometrial tissue 35 days aftertransplantation: a number of nuclei have been immunohistochemicallystained with Ki-67, showing proliferation of glandular and stromal cells(magnification ×400).

Figure 2. Immunohistochemical staining of human CD56.
(A) Pretransplantation endometrial tissue: human CD56-positive cells are present in small numbers(magnification ×400). (B) Day 14 after transplantation: human CD56-positive cells have completely
disappeared from the stroma (magnification ×400). (C) Day 21 after transplantation: human CD56-positive
cells are present in small numbers (magnification×400). (D) Day 28 after transplantation: human CD56positive cells have significantly increased in number in the stroma (magnification ×400).

Other models• Efficacy test model for thrombopoietic drugs
Nakamura, T., Y. Miyakawa, A. Miyamura, A. Yamane, H. Suzuki, M. Ito, Y. Ohnishi, N. Ishiwata, Y. Ikeda,
and N. Tsuruzoe. 2006. A novel nonpeptidyl human c-Mpl activator stimulates human megakaryopoiesis andthrombopoiesis. Blood 107:4300-4307.

NIP-004 is a novel synthetic compound developed to display human thrombopoietin (TPO) receptor (c-Mpl) agonist
activity. NIP-004 displays species specificity, stimulating proliferation or differentiation of human c-Mpl–expressing cells
such as UT-7/TPO and human CD34(+) cells but not murine c-Mpl–expressing cells or cynomolgus monkey cells. To test
the mechanism of its action, we constructed mutant forms of c-Mpl; murine c-MplL490H displayed a response to NIP004, where human c-MplH499L lost this response, indicating that histidine in the transmembrane domain of c-Mpl isessential for its activity. Because histidine is not present in the c-Mpl transmembrane domain of rats, hamsters, rhesus
macaques, and cynomolgus monkeys, we examined the in vivo efficacy of NIP-004 using mice that received
xenotransplants. In immunodeficient nonobese diabetic (NOD)/Shi-scid, IL-2Rgamma(null) (NOG) mice receiving
transplants of umbilical cord blood–derived as CD34(+) cells, NIP-004 increased human megakaryoblasts, maturemegakaryocytes, and circulating human platelets 6-fold, the latter being morphologically and functionallyindistinguishable from normal human platelets. These observations indicate that NIP-004 is a novel human c-Mpl
activator and induces human thrombopoiesis. (Blood. 2006;107: 4300-4307)

Figure 1. NIP-004–induced production of human platelets in NOG mice receiving xenotransplants.
(A) NIP-004 increased the number of circulating human platelets in NOG mice. (B) NIP-004 did not change the number ofmurine platelets or chimerism of HuCD45+ cells. NIP-004 had no effect on the percentage of human B (CD19+) cells, human T(CD3+) cells, and human myeloid (CD33+) cells in the peripheral HuCD45+ cells. Data from panels A-B are expressed as themean ± SEM (n = 3). (C) NIP-004 increased the number of circulating human platelets. The increase was calculated as thenumber of circulating human platelets at individual time points divided by the pretreatment value (day 0). Data are expressed as
the mean ± SEM (n = 3 to 6) or mean ± SD (n = 2). *P < .05, **P < .01 between NIP-004 and vehicle at individual time points.
N.S. indicates no significant differences compared with vehicle.

Figure 2. Morphologic and functional features of human platelets induced byNIP-004 in NOG mice.
(A) Immunoelectron microscopy using antibody against HuCD41a identified human platelets in PRP derived from NIP004–treated mice. The surface of a platelet located in the center is labeled with gold particles (arrow), indicating that it isof human origin. Bar, 1 µm. (B) P selectin (HuCD62p) expression upon ADP stimulation in human platelets was similarly
increased in both vehicle- and NIP-004–treated mice. (C) After stimulation with various concentrations of ADP, there wasa similar dose-dependent escalation in the percentage of PAC-1–positive human platelets from vehicle- and NIP004–treated NOG mice receiving xenotransplants. PAC-1 antibody specifically recognizes the activated form ofGPIIbIIIa. Data are expressed as the mean ± SEM (n = 4).

Other models• Safety test for human cell (ES, iPS, gene-manipulated cells) transplantation
K. Machida, H. Suemizu, K. Kawai, T. Ishikawa, R. Sawada, Y. Ohnishi, and T. Tsuchiya. 2009.
Higher susceptibility of NOG mice to xenotransplanted tumors. J Toxicol Sci 34:123-127.
The purpose of tumorigenicity testing, as applied not only to cell substrates used for viral vaccine manufacture
but also stem cells used for cell-based therapy, is to discriminate between cells that have the capacity to form
tumors and cells that do not. Therefore, tumorigenicity testing is essential in assessing the safety of thesebiological materials. Recently developed NOD/Shi-scid IL2Rgnull (NOG) mice have been shown to be superior
to NOD/Shi-scid (SCID) mice for xenotransplantation of both normal and cancerous cells. To select a suitable
mouse strain as a xenogenic host for tumorigenicity testing, we compared the susceptibility of NOG (T, B, andNK cell-defective), SCID (T and B cell-defective), and the traditionally used nude (T cell-defective) mice totumor formation from xenotransplanted HeLa S3 cells. When 104 HeLa S3 cells were subcutaneously inoculated
into the flanks of these mice, the tumor incidence on day 22 was 10/10 (100%) in NOG, 2/10 (20%) in SCID,and 0/10 (0%) in nude mice. The subcutaneous tumors formed reproducibly and semiquantitatively in a dosedependent manner. Unexpectedly, half of the NOG mice (5/10) that had been inoculated with a mere 101 HeLa
S3 cells formed progressively growing subcutaneous tumors on day 78. We confirmed that the engrafted tumors
originated from inoculated HeLa S3 cells by immunohistochemical staining with anti-HLA antibodies. These
data suggest that NOG mice may be the best choice as a suitable strain for testing tumorigenicity.

Table 1. Comparative growth of HeLa S3 cells among BALB/cA nu/nu, NOD/Shi-scid, and NOG mice.

Cell doseSex(cells/head)
21x101x103

1x104

1x105

Number of mice with tumors (% engraftment)a
BALB/cA nu/nu

NOD/Shi-scidNOGMaleNT

0/5 (0%)3/5 (60%)

FemaleNT

0/5 (0%)3/5 (60%)

Total

NT

0/10 (0%)

6/10 (60%) *Male

0/5 (0%)0/5 (0%)3/5 (60%)

Female0/5 (0%)0/5 (0%)3/5 (60%)

Total

0/10 (0%)

0/10 (0%)

6/10 (60%) #Male

0/5 (0%)2/5 (40%)

5/5 (100%) **Female0/5 (0%)0/5 (0%)5/5 (100%) ##Total

0/10 (0%)

2/10 (20%)

10/10 (100%) ##Male

5/5 (100%)

5/5 (100%)

NTFemale

3/5 (60%)

4/5 (80%)

NT

Total

8/10 (80%)

9/10 (90%)

NTa Engraftment was evaluated 22 days after inoculation by 1 x 103, 104 , and 105 cancer cells, and 43 days after

inoculation by 1 x 102 cancer cells. Fisher's exact test was performed in statistical analysis. * P < 0.05 compared tothe NOD/Shi-scid strain. ** P < 0.01 compared to the BALB/cA nu/nu strain. # P < 0.05 compared to theBALB/cA nu/nu and NOD/Shi-scid strain. ## P < 0.01 compared to the BALB/cA nu/nu and NOD/Shi-scid strain.
NT: not tested.