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1 Lab. Anim. Res. 2010: 26(2), Safety Evaluation of Human Fibroblasts in Mice: Tumorigenicity, 13-week Toxicity and Distribution Studies Hyung Jun Choi 1,2, Euna Kwon 1, Jeong-Hee Sohn 1, Jeong-Hwan Che 1, Kook Hyun Lee 1, Jong Wan Kim 3, Jaydo Choi 3 and Byeong-Cheol Kang 1,2 * 1 Department of Experimental Animal Research, Clinical Research Institute, Seoul National University Hospital, Seoul, Korea 2 Department of Immunology and Laboratory Animal Medicine, College of Medicine, Seoul National University, Seoul, Korea 3 S.BIOMEDICS Co., Ltd. Seoul, Korea Human fibroblasts were developed for cellular therapy with the aim of correcting of depressed scars, but the safety of that in vivo is unclear. In this study, we assessed the safety of human fibroblasts by investigating the tumorigenicity, 13-week toxicity and through distribution studies. In the tumorigenicity test, nude mice were divided into three dosage level treatment groups with a negative/positive control group. At 6 months after intradermal transplantation, all of the treatment groups showed no development of a nodule on the injection sites and organs. Toxicity studies were performed using ICR and BALB/c mice for 13 weeks. The mice were divided into three dosage level treatment groups with a control and a syngeneic group. There was no treatment-related effect on clinical signs, mortality, body weight, food/ water consumption, hematology, serum biochemistry, urine, necropsy findings and histopathological findings in any groups. These results suggest that the no-observed-effect level (NOEL) of the human fibroblasts was greater than cells/kg for mice. In the distribution study, groups were treated with fibroblasts labeled with a fluorescent dye (CM-DiI) at low and high doses with a control and a syngeneic group. At 24 hours, a large percentage of the labeled fibroblasts were observed at the dermal layer. At 3 months, fluorescence of the labeled fibroblasts continued to be observed. Other tissues were not detected the fluorescence at any time. These studies demonstrate that the safety of human fibroblasts is reasonable with no toxic effect, no tumorigenicity and retention in the dermis. Our studies define preclinical safety testing standards relevant to the development of cellular therapeutics. Key words: Fibroblast, tumorigenicity, toxicity, distribution, cell therapy Received 26 November 2009; Revised version received 19 March 2010; Accepted 14 June 2010 se s» j» w ƒ(autologus), (allogenic), (xenogenic) s, w ù kw s w p y j w mw e, t w. v se v v w» s w yù w w v {l e w se. s(fibroblast) (Boss et al., 2000) v w» s(adult stem cell) v w *Corresponding author: Byeong-Cheol Kang, Department of Experimental Animal Research, Clinical Research Institute, Seoul National University Hospital, 28 Yongon-dong, Jongno-gu, Seoul , Korea Tel: Fax: bckang@snu.ac.kr w w v s yw v g. p ƒ s e š v ù. s w x(guerret et al., 2003) x(watson et al., 1999; Boss et al., 2000) v z ùkü š» y wš y. Apligraft (Guerret et al., 2003)ù Orcel v se ƒ FDA (Food and Drug Administration) q, ü ƒ se Holoderm (Lee et al., 2006), se Kaloderm (Seo et al., 2007; Yoon et al., 2008) t t xƒ. w» s w se, x x mw y w v. 181

2 182 Hyung Jun Choi et al. t» t [ t x ] sƒ», yw se w» w š. p» s w se sƒ w x t z sƒ w ƒ z w w. x w ( ) š w mw sƒƒ w. w s w, Galbraith (2004) s w s w w ³ w.» s ƒ (selfrenewal) (proliferative potential) w s w ƒ (Rubio et al., 2005; Trosko et al., 2006) š ƒ x w w. yw t w 2 x (carcinogenicity) ww, se v ù SCID (severe combined immunodefieicency) w 6 x(tumorigenicity) (Lawrenz et al., 2004; Lee et al., 2007) ww. s z w dw» w, k, x yw, x w v q l w w, t w y w y w w. w w w w sxk y y w» w w s s w» ƒ v w.» s w x (Lee et al., 2007), (Liu et al., 2006; Kovacsovics- Bankowski et al., 2008; Vilalta et al., 2008) s x(cho et al., 2002; Allers et al., 2004; Benten et al., 2006; Meyerrose et al., 2007) t š, v se s w w w. x s x, s sƒw» w, nude mouse w in vivo x, ICR mouse w 13 x, ICR mouse CM-DiI x Ÿ w ü s x w. l x v se s (human fibroblasts) y v w k z y v v ƒ w z wš. x S.BIOMEDICS Co., Ltd. (Seoul, Korea) s ww w s s w s, sw,, ³,, gv x w q s n syk k œ, n n s w y w z w. wr, 13 x s x s x w y w s ƒw» w, s wš (BALB/c) w k s œ w. n ƒ x viability test w. x Ringer s Solution( ( ), Seoul, Korea) w. s x, ü s sƒw» w s CellTracker TM CM-DiI (Molecular Probe inc., Eugene, Oregon, United States) w xÿt w. xÿt s CM-DiI (2 µg/ml) ƒw z 37 o C 5 ew z, 4 o C 15 ew w. lò x 7 f Nude mouse (BALB/c-nu) Harlan (Indianapolis, United States) l w š, 13 x 7 ICR mouse BALB/c mouse ( ) p( û, w ) l w, s x 7 f ICR mouse BALB/c mouse ( ) p( û, w ) l w. w w x y» w x w. x y 22±2 o C, 50±10%, y»z 12~18z/hr, 12, 150~300 Lux, 55 db w w.» Individually Ventilated Caging System (IVCS) polysulfone f 5 w 1z ³f yw. ³ x šx (u ù x 5057) w, š š ³(autoclave)w w œ w. x w w (AAALAC» ) IACUC National Research Council x ƒ w. l l x: x s

3 1 (60 kg) cells, s w, wš, œ 10 w 3 x n ( , , cells/kg), w (A431 cell). 10 w v x n w. z š w v ü 2 1z 3z w. d w m n (0.05 ml/30 g B.W.) y w n w. 13 x: x n w 3 x n ( , , cells/kg), s s w» w BALB/c syngeneic group ( cells/kg). ƒ ƒƒ 20 w. v x n w. z v ü 1z 4 4z w. d w m n (0.05 ml/30 g B.W.) y w n w. s x: ( 1 (60 kg) cells) 13 x n š w wš, œ 100 w 2 x n ( , cells/kg). s w w» w BALB/c mouse ( cells/kg) w w w. s s w» w n z 24, 4, 13, 6 w., z n 13 x w w. á x: x n w w 1 2z, z w š, w 1 1z» w. w x, z x ¾ 2z d wš, d w. y z x ¾ 2 z y w, j» calipers w d w (π/6 ¼ s ) œ w. SNUH- IACUC «š 25 mm w. x w x w isoflurane w w Safety of human fibroblasts in mice 183 xw z» w w.» d x w,, s, ( Á ), ( Á ), ù ( Á ),, w d w š, w ( ) w. 13 x: x Õ t x» Ö ( t t š ) w w. d x w w. f z l x ¾ 1z d w. x» z 13 ƒ ƒƒ 5 w w, nÿ ƒƒ w w w š, metabolic cage w w z, x (Combur10Test M, Roche, Germany) w urine analyzer (Miditron Junior II, Roche, Germany) ph, specific gravity, leukocyte, nitrite, protein, ketone body, urobilinogen, bilirubin, glucose, x(occult blood) w. x w x yw w x w. Á ƒƒ x yw, 5 x w, 5 x š w. x w white blood cell (WBC), red blood cell (RBC), hemoglobin (Hb), hematocrit (HCT), platelet (PLT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), leukocyte differential Animal Blood Counter (Melet schloesing laboratoires, MS 9-5, France) w w. x yw w x 30 ew š z (3,000 rpm, 15 min)w x w blood urea nitrogen (BUN), cholesterol (CHOL), total protein (TP), albumin (ALB), total bilirubin (TB), alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CRN), triglycerides (TG), glucose, K, Cl, Ca, P, Na, A/G ratio automatic chemistry analyzer (HITACHI, 7070, Japan) d w. x š prothrombin time (PT; turbidimetric method), partial thromboplastin time (PTT; turbidimetric method) coagulation Analyzer (ACL-100, Lexington, United States) w d w. Á ƒƒ w» d w w, ù 10 w w.

4 184 Hyung Jun Choi et al. v, t w z vw vw k w. z, { š ü» k w.»á, v,, v, k, ({, n ), {,», s»,,, x,,,,,,,,,, Ÿ, û,, šy, šy, ù, ª,,, w,,»,»k».» d x w,, s, {,, ( Á ), ( Á ), šy( Á ), ù ( Á ),, w d w š, w ( ) w. w d óù» šy Bouin, Davidson,»k» 10% s 1 w š» n, {, kz, x» w q v s» s w j m 2~3 µm r š hematoxylin & eosin (H&E) w. w w w. s x: d x 13 x w. x ü s sƒw» w s xÿt (CellTracker TM CM-DiI, Molecular Probe Inc, Eugene, United States) w, z xÿx (Olympus IX71, Olympus Otical Co., Japan; TRITC filter) w. x n z 24, 4, 13 w,, liver, lung, heart, thymus, kidney (left/right), adrenal gland (left/ right), spleen 10% s š w w. ƒ» 10 serial sections w. x d e SPSS (SPSS 12.0K) w ANOVA w z α=0.05 w š, Tukey-Kramer test Dunnet t-test ww ƒ m w w (P<0.05). ICR mouse BALB/c mouse data strain š w m w. Figure 1. Tumor incidence rate of female BALB/c-nu mice intradermally treated with human fibroblasts in the tumorigenicity study. l : x» x w. w 1 q w, n n ƒ 1 ƒvƒ x w ù œ w. x w š, w. y: x w x» w w. (Figure 1): x z 27 2 z w x e,.» : z» w w n w w (P<0.05). q : s cells/kg B.W. w w.

5 Safety of human fibroblasts in mice 185 Figure 2. Body weight changes of male (left chart) and female (right chart) mice intradermally treated with human fibroblasts in the 13-week toxicity study. Ð l : x» x w. w f n 1 n 2 l 14 ¾, 1 n 12 l 14 ¾» ƒvƒ x w ù f œ w. x w š, w. y(figure 2): x w x» w w., z : f š n n 2 (4.57±0.219 vs 3.94±0.302 g) w w ƒ (P<0.05). f x» w w. z f n 2 š n (0.05 vs 0.02 g) w ƒ š, f n 5 n š n (0.00 vs 0.05 vs 0.06 g) w ƒƒ. n 6 n (0.04 vs 0.02 g) w ƒ. f n 2 š n ( vs g) w ƒƒ. f w w. : x n w x» w w. x w (Table 1): f w w yƒ, f n leukocytes w ( /µl vs /µl) 3 w ƒƒ, eosinophils w (3.0 vs 1.1%) w ƒ. š n MCHC w (34.0 vs 31.3 g/dl) w ƒ (P<0.05). x yw (Table 2): f š n ALT w (25 IU/L vs 17 IU/L) w ƒ, n š n Cl w (109.3 vs vs mmol/l) w ƒƒ, š n Na w (149.9 vs mmol/l) w ƒƒ. f š n TP w (4.8 vs 5.0 g/dl) w ƒƒ, n n CRE w (0.47 vs 0.39 vs 0.40 mg/dl) w ƒ, n š n K w (4.83 vs 5.29 vs 5.33 mmol/l) w ƒƒ. : 13 w, w p w y w, x w p w y w. x š : x š w x n w w w. (Table 3): f n 2

6 186 Hyung Jun Choi et al. Table 1. Summary of the hematological values of mice intradermally treated with human fibroblasts in the 13-week toxicity study le a M le a m e F Dosage in (cells/kg B.W.) Items 0 (Control) (Syngeneic) Leukocytes (10 3 /µl) 3.32± ± ± ± ±0.412 Erythrocytes (10 6 /µl) 7.8± ± ± ± ±0.62 Hemoglobin (g/dl) 11.2± ± ± ± ±1.03 Hematocrit (%) 33.7± ± ± ± ±3.11 Platelets (10 3 /µl) 953± ± ± ± ±131.5 MCV (fl) 43.6± ± ± ± ±0.63 MCH (pg) 14.4± ± ± ± ±0.54 MCHC (g/dl) 33.2± ± ± ± ±0.80 Neutrophils (%) 6.7± ± ± ± ±5.73 Eosinophils (%) 3.1± ± ± ± ±2.80 Basophils (%) 0.4± ± ± ± ±0.09 Lymphocytes (%) 83.5± ± ± ± ±4.19 Monocytes (%) 4.7± ± ± ± ±1.25 Leukocytes (10 3 /µl) 2.67± a ± ± ± ±1.482 Erythrocytes (10 6 /µl) 7.7± ± ± ± ±0.32 Hemoglobin (g/dl) 11.2± ± ± ± ±0.53 Hematocrit (%) 32.9± ± ± ± ±1.09 Platelets (10 3 /µl) 896± ± ± ± ±28.6 MCV (fl) 42.7± ± ± ± ±0.62 MCH (pg) 14.5± ± ± ± ±0.11 MCHC (g/dl) 34.0± ± ± a ± ±0.64 Neutrophils (%) 8.4± ± ± ± ±1.81 Eosinophils (%) 3.0± a ± ± ± ±1.21 Basophils (%) 0.3± ± ± ± ±0.11 Lymphocytes (%) 81.0± ± ± ± ±2.15 Monocytes (%) 5.5± ± ± ± ±0.91 All values are expressed as mean±sd, n=5 a Significantly different from the control group; P<0.05.» ƒvƒ, v w cyst 1, Ét cyst 1 ƒ. f û / š 1, cyst 1, Ét cyst 1, 1 ƒ. f n ªƒ cyst 1, ù cyst 1 ƒ, n ù cyst 1 ƒ. p w w.» (Table 4 and Table 5): z» w d w, f d n ( vs g) w ƒ, f d š n ( vs g) w ƒ š, d š n ( vs g) w ƒ. ( vs vs g) n š n w ƒ. w, f d n ( vs g%) w ƒ, n ( vs g%) w ƒ. f n n ( vs vs g%) w ƒ, n, n, š n ( vs vs vs g%) w ƒ. d ( vs , vs g%) š n ( vs , vs g%) w ƒ, n n ( vs vs g%) w w. w (Table 6 and Figure 3): w š w ù,» p. w

7 Safety of human fibroblasts in mice 187 Table 2. Summary of the serum biochemical values of mice intradermally treated with human fibroblasts in the 13-week toxicity study le a M le a m e F Dosage in (cells/kg B.W.) Items 0 (Control) (Syngeneic) Urea Nitrogen (mg/dl) 27.9± ± ± ± ±4.36 Cholesterol (mg/dl) 145± ± ± ± ±14.8 Total Protein (g/dl) 4.7± ± ± ± ±0.10 Albumin (g/dl) 1.5± ± ± ± ±0.03 Total Bilirubin (mg/dl) 0.1± ± ± ± ±0.04 Alkaline Phosphatase (IU/L) 37± ± ± ± ±6.00 Aspartate Aminotransferase (IU/L) 54± ± ± ± ±29.9 Alanine Aminotransferase (IU/L) 25± ± ± a ± ±9.70 Creatinine (mg/dl) 0.5± ± ± ± ±0.05 Triglycerides (mg/dl) 67± ± ± ± ±27.7 Glucose (mg/l) 173± ± ± ± ±15.8 A/G ratio 0.5± ± ± ± ±0.00 Potassium (mmol/l) 5.65± ± ± ± ±0.355 Chlorine (mmol/l) 109.3± ± a ± a ± ±1.15 Calcium (mg/dl) 8.3± ± ± ± ±0.23 Phosphorus (mg/dl) 6.5± ± ± ± ±0.76 Sodium (mmol/l) 149.9± ± ± a ± ±1.12 Urea Nitrogen (mg/dl) 23.3± ± ± ± ±4.47 Cholesterol (mg/dl) 90± ± ± ± ±8.00 Total Protein (g/dl) 4.8± ± ± a ± ±0.15 Albumin (g/dl) 1.6± ± ± ± ±0.08 Total Bilirubin (mg/dl) 0.0± ± ± ± ±0.03 Alkaline Phosphatase (IU/L) 66± ± ± ± ±4.7 Aspartate Aminotransferase (IU/L) 60± ± ± ± ±28.2 Alanine Aminotransferase (IU/L) 18± ± ± ± ±19.1 Creatinine (mg/dl) 0.47± a ± a ± ± ±0.13 Triglycerides (mg/dl) 67± ± ± ± ±29.7 Glucose (mg/l) 182± ± ± ± ±35.7 A/G ratio 0.5± ± ± ± ±0.00 Potassium (mmol/l) 4.83± ± a ± a ± ±0.369 Chlorine (mmol/l) 115.0± ± ± ± ±1.48 Calcium (mg/dl) 8.1± ± ± ± ±0.21 Phosphorus (mg/dl) 6.4± ± ± ± ±0.83 Sodium (mmol/l) 150.0± ± ± ± ±1.90 All values are expressed as mean±sd, n=10 a Significantly different from the control group; P<0.05. p ùkü., x x w w w š q. f n 1 ª k (deciduoma) ù n q (Figure 3A). x n y n (injection site). š ( cells/kg) n w ICR BALB/c f n (foreign body granulomatous inflammation). v s s š,, (needle-like crystals) xk (Figure 3B). n x q. f BALB/c œs(centrilobular vacuolation) ƒ ƒ f BALB/c q (Figure 3C). q : ww, s ICR mouse 13 x w (NOEL) cells/kg. g l : x» x

8 188 Hyung Jun Choi et al. Table 3. Summary incidence of the gross findings of mice intradermally treated with human fibroblasts in the 13-week toxicity study Sex Male Female Dosage in (cells/kg B.W.) 0 (Control) (Syngeneic)(Control) Tissue/Observation Skin scab around genitalia Seminal vesicle/coagulation gland atrophy Kidney cyst atrophy Preputial gland cyst Mammary gland subcutaneous, mass Uterus uterine horn, cyst Ovary cyst (Syngeneic) w. w x w š w. y: y x w x» w w. 12 w ù, œ w x w w, œ z z y w. : p w w. ü s sƒ(figure 4): s ü s w 24, 4, 13 ¾. 13 n, Syngeneic, š n w.» x s w. q : x w s 13 ¾ w y w,» s y. v se ù {l,, y ù e ƒ ù x ù ƒ e w ƒ y. w ü se y w š. se w x x sƒ v. ù» s p š w w sƒ ƒ y š. se s sƒw» w, 13 š s x w. se x Lawrenz et al. (2004) w š š w, A375 cell line š x w. n ( )n w v ü 2 1z 3z w. s BALB/c-nu mouse w x s cells/kg B.W. w w. x mw se sƒ w x v mg wš w š, s x x y w y w. w x, s» s x» s mw w y w (Lee et al., 2007). m se ƒ e, z {w» w sƒ n, w ƒ w. x mw (Liu et al., 2006; Kovacsovics-Bankowski et al., 2008; Vilata

9 Safety of human fibroblasts in mice 189 Table 4. Summary of the absolute organ weight values of mice intradermally treated with human fibroblasts in the 13-week toxicity study ¾ le a M le a m e F Dosage in (cells/kg B.W.) Items 0 (Control) x10 7 (Syngeneic) Necropsy B.W ± ± ± ± ± Liver ± ± ± ± ± Spleen ± ± ± ± ± Kidney (R) ± ± ± ± ± Kidney (L) ± ± ± ± ± Adrenal gland (R) ± ± a ± a ± ± Adrenal gland (L) ± ± ± ± ± Testis (R) ± ± ± ± ± Testis (L) ± ± ± ± ± Thymus ± ± ± ± ± Heart ± ± ± ± ± Lung ± ± ± ± ± Brain ± ± ± ± ± Pituitary gland ± ± ± ± ± Necropsy B.W ± ± ± ± ± Liver ± ± ± ± ± Spleen ± ± ± ± ± Kidney (R) ± ± ± a ± a ± Kidney (L) ± ± ± a ± a ± Adrenal gland (R) ± ± ± ± ± Adrenal gland (L) ± ± ± ± ± Ovary (R) ± ± ± ± ± Ovary (L) ± ± ± ± ± Thymus ± ± ± ± ± Heart ± ± ± ± ± Lung ± ± ± ± ± Brain ± ± a ± a a ± a ± Pituitary gland ± ± ± ± ± All values are expressed as mean±sd, n=10 a Significantly different from the control group; P<0.05. et al., 2008) e s w sƒ w, t x» w w š. x x, n,» w t w w w. x, n, n, š w se ww sƒw w w. ICR mouse 13 x n ( cells/kg B.W.) w š, œ 10 n ( cells/kg B.W.), š n ( cells/kg B.W.), w v x (Ringer s solution) n w ƒ ƒƒ 20 w. s w w w» w BALB/c mouse w s vü n w w w. x n 1z 4 4 z n w. x», f n 2» ƒvƒ ù œ w, x w ù p. y w w y., x w x yw,, yƒ ù, y š y x w y.» f,, f,,, w w ù w.» w ù, x n w. w, w x n w

10 190 Hyung Jun Choi et al. Table 5. Summary of the relative organ weight values of mice intradermally treated with human fibroblasts in the 13-week toxicity study le a M le a m e F Dosage in (cells/kg B.W.) Items 0 (Control) (Syngeneic) Liver ± ± ± ± ± Spleen ± ± ± ± ± Kidney (R) ± ± ± ± ± Kidney (L) ± ± ± ± ± Adrenal gl. (R) ± ± a ± a ± ± Adrenal gl. (L) ± ± ± ± ± Testis (R) ± ± ± ± ± Testis (L) ± ± ± ± ± Thymus ± ± ± ± ± Heart ± ± ± ± ± Lung ± ± ± ± ± Brain ± ± a ± a ± ± Pitui gl ± ± ± ± ± Liver ± a ± a a ± a a ± a ± Spleen ± a ± a a ± a ± ± Kidney (R) ± ± a ± a a ± a ± Kidney (L) ± ± a ± a a ± a ± Adrenal gl. (R) ± ± ± ± ± Adrenal gl. (L) ± ± ± ± ± Ovary (R) ± ± ± ± ± Ovary (L) ± ± ± ± ± Thymus ± ± ± ± ± Heart ± ± ± ± ± Lung ± ± ± ± ± Brain ± a ± a a ± a ± ± Pitui gl ± ± ± ± ± All values are expressed as mean±sd, n=10 a Significantly different from the control group; P< s w w w» w BABL/c mouse w s vü n w w w, m (ICR vs BALB/c) ù x n w w y. s ICR mouse 13 x, s w (NOEL) cells/kg B.W. q. w s ù w y,» y w s x sƒw. se s x w s dye w xÿ x mw y w (Hebda et al., 1999; Ferrari et al., 2001; Moeller et al., 2003; Sandulache et al., 2003), wš e w (Allers et al., 2004; Thompson et al., 2005), enhanced green fluorescent protein (egfp) x y w (Meyerrose et al., 2007), s p gene mw PCR (Vilata et al., 2008) in situ hybridization (Cho et al., 2002; Benten et al., 2006), w bioluminesence imaging(vilata et 2008), MRI (Bulte et al., 2002)ù PET (Adonai et al., 2002) y w w mw š. p s s x mö CM-DiI s w ƒ,, s v z w x 24» sƒ w xÿx m w w x (Hebda et al., 1999; Sandulache et al., 2003). s ü s sƒw» w w ICR mouse w s x s xÿt (CellTracker CM-DiI) w TM, z xÿx w w. šƒ ƒ v s w, w w. ( cells/60kg) 13 x n š w n ( cells/kg B.W.) š n ( cells/kg B.W.), w v x (Ringer s solution) n w al.,

11 Safety of human fibroblasts in mice 191 Table 6. Summary incidence of the microscopic observations of mice intradermally treated with human fibroblasts in the 13-week toxicity study ¾Dosage in (cells/kg B.W.) Tissue/Observation Male Female 0 (Control) (syngeneic) (Control) (syngeneic) Liver Glycogen vacuolation Centrilobular vacuolation Parenchymal inflam cell foci Eosinophilic focus Kidneys Number examined Cortical cyst Pelvic dilatation Cortical vacuolation Nephropathy Subtransi. epi. inflam. infil Interstitial inflam cells Tubular cast(s) Tubular basophilia Tubular hypertrophy, focal Adrenal glands Number examined Cortical inflammatory cells Pigmentation Subcapsular cell focus Urinary bladder Number examined Transi epi-subepi inflam cells Spleen Haemopoiesis Pancreas No abnormalities detected Thymus Number examined Involution/atrophy Thyroid Number examined Ectopic thymus Follicular distension Parathyroid glands Number examined Lymphoid cells Trachea Number examined Imflam cell debris in lumen Subepithelial inflam cells Oesophagus Number examined Submucosal inflammatory cells Tongue Mast cells

12 192 Hyung Jun Choi et al. Table 6. Continued ¾Dosage in (cells/kg B.W.) Tissue/Observation Male Female 0 (Control) (syngeneic) (Control) (syngeneic) Lung Perivascular inflam cells Alveolar macrophages Osseous metaplasia Heart Myocardial inflam cells Epicardial inflammatory cells Salivary gland Perivascular inflam cells Cervical lymph nodes No abnormalities detected Forestomach Submucosal inflammatory cells Epithelial inflam cells Glandular stomach Glandular cyst(s) Submucosal inflammatory cells Epithelial hyperplasia Small intestine No abnormalities detected Large intestine No abnormalities detected Mesenteric lymph nodes No abnormalities detected Preputial/Clitoral glands Number examined Inflammation Skin/mammary gland Number examined Scab Pannicu muscle-inflam cells Epidermal hyperplasia Deep dermal inflam cells Pannicu muscle-degeneration Subcutaneous granuloma Eye No abnormalities detected Haderian gland Inflammatory cell infiltration

13 Safety of human fibroblasts in mice 193 Table 6. Continued ¾Dosage in (cells/kg B.W.) Tissue/Observation Male Female 0 (Control) (syngeneic) (Control) (syngeneic) Brain No abnormalities detected Pituitary Number examined Cyst Femur/Bone marrow Granulocyte hyperplasia Nasal turbinates Olf epi-eosinophilic inclusion Inflam cell debris in lumen Olf epi-inflammatory cells Olf epi-subepi inflam cells Resp epi-inflammatory cells Resp epi-subepi inflam cells Resp epi-eosinophilic inclusio Transi epi-inflam cells Transi epi-subepi inflam cells Resp epi-erosin Resp epi-hyperplasia Sternum+marrow Granulocyte hyperplasia Sciatic nerve Number examined Perivascular inflam cells Testis Number examined Seminiferous tubular degenerat Seminiferous tubular vacuolati Rete tubular hyperplasia Epididymis Number examined Sperm granuloma Degenerate germ cells in lumen Prostate Number examined No abnormalities detected Seminal vesicle Number examined No abnormalities detected Ovary Number examined Cyst Follicular cyst Prominent corpus leuteum Endometriosis

14 194 Hyung Jun Choi et al. Table 6. Continued ¾Dosage in (cells/kg B.W.) Tissue/Observation Male Female 0 (Control) (syngeneic) (Control) (syngeneic) Uterus Number examined Cystic glands Luminal dilatation Deciduoma Vagina Number examined Inflam cell debris in lumen Injection site Scab Pannicu muscle-regeneration Pannicu muscle-inflam cells Epidermal hyperplasia Deep dermal inflam cells Pannicu muscle fibrosis moderate Pannicu muscle-degeneration Deep derm foreign body granulo Subcutaneous inflam cells z w v ü 1z 4 4z w. s w w w» w BALB/c mouse w s vü n w w w, n z 24, 4, 13 w s sƒw. ü s w, 24 n, š n vü w sƒ w xw xÿx mw y w. xÿ n w s w. 4 w sƒ 24 w xÿ ù kù, vü. 13 w w w xÿ vü w. xÿ s xÿ (Hebda et al., 1999; Sandulache et al., 2003). BALB/c mouse s s x s š n w. s s vü s w ƒ. x(schrepfer et al., 2007; Vilalta et al., 2008; Fischer et al., 2009) w n s, x». n w, ü n y mw s x x sƒ, vün w w w sƒ w š. Figure 3. Microscopic findings of ICR mice intradermally treated with human fibroblasts in the 13-week toxicity study. (A) Deciduoma in uterus. 40, (B) Granulomatous inflammation in the dermis. Note the needle-like crystals and necrosis. 40, (C) Centrilobular vacuolation in liver. 40. H&E.

15 Safety of human fibroblasts in mice 195 Figure 4. Photomicrographs of the skin and other organs of mice Intradermally treated with labeled human fibroblasts in the distribution study. The images from the first layer to the third layer are each skin sample at 24 hours, 4 weeks and 13 weeks ( 100). The images of the fourth layer are other organs that were not detected with the labeled fibroblasts ( 40). se s n w ƒ(autologous) (allogeneic) ù, se x sƒ ww, (xenogeneic transplantation)., w w š dw, x x k w. x BALB/c mouse w s (BALB/c) (syngeneic transplantation)w, s ICR mouse w w w w w w. w, v, SCID, transgenic w w yw w y w x š w w. x w s cells/kg B.W. w w, w (NOEL) cells/kg B.W. q š, 13 ¾ w,» s y. mw x, x š s x se w š, w sƒ l se sƒ t y y» w». k Adonai, N., Nguyen, K.N., Walsh, J., Iyer, M., Toyokuni, T., Phelps, M.E., McCarthy, T., McCarthy, D.W. and Gambhir, S.S. (2002) Ex vivo cell labeling with 64Cu-pyruvaldehydebis(N4-methylthiosemicarbazone) for imaging cell trafficking in mice with positron-emission tomography. Proc. Natl. Acad. Sci. U.S.A. 99(5), Allers, C., Sierralta, W.D., Neubauer, S., Rivera, F., Minguell, J.J. and Conget, P.A. (2004) Dynamic of distribution of human bone marrow-derived mesenchymal stem cells after transplantation into adult unconditioned mice. Transplantation 78(4), Benten, D., Cheng, K. and Gupta, S. (2006) Identification of transplanted human cells in animal tissues. Methods Mol. 326, Boss, W.K., Jr., Usal, H., Chernoff, G., Keller, G.S., Lask, G.P. and

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