Fibrous formation observed at the implantation site ofundifferentiated iPS cells in NOG mice

P-64

NOGマウスにおける未分化iPS細胞の移植部位 (背部皮下) に認められた線維形成
Takafumi Oshikata (押方孝文)1, Masashi Iida (飯田真志)2, Koshiro Katoku (可徳小四郎)1, Takeshi Kanno (菅野剛)1
1: LSIM Safety Institute Corporation Pathology Department (LSIM安全科学研究所 病理研究部)
2: LSIM Safety Institute Corporation Safety Assessment Department (LSIM安全科学研究所 安全性研究部)

Background NOG mice were subcutaneously implanted with iPS cells and HeLa cells into the dorsal tissue
for evaluation of the minimum tumor-producing dose and tumorigenic period.

We examined histopathologically whether
this fibrous formation was derived from
iPS cells.

 Tumor formation was observed in the group implanted with HeLa cells. On the other hand,no tumor formation but fibrous formation was observed in the group implanted with iPS cells.

MethodGroup design
Study outline

 Inoculation of cell cultures:○ Human adipose-derived mesenchymal stem cells (hMSC), Lot No. 0000672320,
Lonza KK
○ HeLa cells, Lot No. 61647128, ATCC
○ Human undifferentiated iPS cells (iPS cells), Lot No. P11025, Takara Bio Inc.

Inoculation of Dose level Concentration Dose volume
Number ofGroupSex
cell cultures (cells/body)animal(cells/mL)(μL/body)
67ControlhMSC
71 × 101 × 101107
Test article 1 HeLa cells
10100
7100
Male100
1000
71107
Test article 2
iPS cells
10100
7100
1000
7 Age at implantation: 7 weeks

Observation period

 Animal: Mouse, NOD/Shi-scid, IL-2RγKO Jic (NOG) Implantation site: Dorsal subcutaneous tissue

 Collection and examination: All organs (including implantation site) Tissue fixation: 10 vol% phosphate buffered formalin solution

Day 1

Day 113

Day 141

Implantation DateCollection1

Collection2

 Stain:All organs
Hematoxylin and eosin stain (HE)

1: Control and test article 1 groups
2: Test article 2 groups(Since no tumor formation was observed, the observation period was extended beyond day 113.)

Implantation site in the test article 2 groups
Masson trichrome stain (MT), Anti-human lamin stain (anti-lamin)

We report mainly on the Implantation site of Test article 2 groups.

ResultTest article 1
Tumor formation (HeLa cells 1×100 cells/body group)

Macroscopic Examination

HEGroup
Inoculation of cell cultures

Control

Test article 1

Test article 2

hMSC

HeLa cells

iPS cells

Dose level (cells/body)110100110100

Number of animal

1×106
7777777Mass, subcutaneous0017000Nodule, subcutaneous

03

50003

Microscopic Examination (HE stain)
Group
Inoculation of cell cultures

Control

Test article 1

Test article 2hMSC

HeLa cells

iPS cells

Dose level (cells/body)

1×10
7

Number of animalLamin

HE6110100

110100777777Tumor formation, subcutis

0367000

Fibrous formation, subcutis

0000124※ : Findings of other organs are not shown.

※:The tumor cells were derived from HeLa cells, as they
were positive for anti-lamin staining.

Test article 2HEControl

Lamin
Control

iPS cells 1×100 cells/body group

iPS cells 1×100 cells/body group▲; Positive, ▲; Negative

MTControl

Summary

iPS cells 1×100 cells/body group Necropsy revealed white nodules at the implantation site in the100 cells/body group.
 Histopathological examination revealed fibrous formation at theimplantation site in all groups.
 The fibers consisted of mature collagen fibers with spindle-shapednuclei, which stained blue by MT.
 Adipocytes were observed interspersed among collagen fibers.
 Anti-lamin staining showed positive in nuclei of collagen fibers andadipocytes, but negative in some nuclei.

COI Disclosure Information
Takafumi Oshikata
In connection with this presentation, there is no COI to be disclosed with any companies.
本演題に関連して開示すべき利益相反はありません。

Discussion Anti-lamin staining showed positive in nuclei of collagen fibers and adipocytes,

but negative in some nuclei, suggesting that fibrous formation (with adipocytes)
was formed from cells derived from mice and humans.
 These results suggest that when iPS cells are implanted into immunodeficientanimals, iPS cells differentiate into mature tissues and grow, and those iPS cells
form into mature tissues by engulfing the host-derived cells other than iPS cells.