Nod scid mice

10⁶ treated hESC-CMs re-suspended in Matrigel (354234, BD Biosciences) were injected subcutaneously between the scapulars of immunocompromised NOD-SCID mice that were purchased from Envigo (Israel). Untreated hESC-CMs as well as undifferentiated hESCs were injected as well, for reference. Between 9–11 weeks after injection, incidences of teratomas were identified by MRI body scan and evaluated by H&E staining. MRI was performed using the M7 1-Tesla compact ICON system (Aspect Imaging, M7, Israel), equipped with a set of application-specific radiofrequency (RF) 80-mm mouse body coils. For in vivo imaging, animals were maintained in an anesthetized state with 1.5% isoflurane in O₂ and placed on a specially designed heated bed. MRI acquisition parameters included fast spin echo with a repetition time of 2,500 ms and echo time of 74 ms. Fifteen axial slices of 0.25 mm with a gap of 0.1 and a matrix of 256 × 256, field of view of 40 mm and acquisition time of 7.4 min. were collected. Dissected tissues were fixed in 4% (v/v) PBS-buffered formalin, gradually dehydrated in alcohol solutions (70–100%), embedded in paraffin and sectioned into 5 µm thick slices. Serial cross-sections were stained with hematoxylin-eosin (H&E) to detect structures that resemble all three germ layers.

Female 5 to 6 week-old non-obese diabetic severely compromised immunodeficient (NOD/SCID) mice were obtained from Beijing HEK Bioscience. K562-R cells (1 × 10⁷) were implanted with matrigel (BD Biosciences) subcutaneously into the right flank of sub-lethally irradiated (250 cGy) NOD/SCID mice. All mice were randomized into two groups, namely, a vehicle control group and a treatment group (n = 4 per group). Mice were treated with CAY10683 (50 mg kg⁻¹; intraperitoneally), IM (50 mg kg⁻¹; intraperitoneally), or with both agents daily for 21 days. Tumor volume was measured twice per week with calipers and was calculated as tumor volume (mm³) = L × W²/2 (L represents the largest diameter and W is the smallest diameter of the tumor). The weights of the mice were also monitored. The survival times of the mice were recorded and analyzed. All animal experiments were approved by the Ethics Committee of Guizhou Medical University.

NOD.CB17-Prkdcscid/J (NOD/SCID) male mice were purchased from Charles River Laboratories, Inc. (Wilmington, MA, USA). Mice were housed under specific pathogen-free conditions with a 12 h light/dark cycle and provided ad libitum access to tap water and food. Huh7 cells (approximately 1.0 × 10⁶ cells) were resuspended in 200 μL of a 1:1 DMEM:Matrigel (BD Biosciences) mixture with control IgG (n = 3, 100 mg/10⁶ cells) or anti-DKK-1 neutralizing antibody (n = 3, 100 mg/10⁶ cells) and subcutaneously injected into 6-week-old NOD/SCID mice. Mice were euthanized, and the tumor volume was evaluated on day 28 after xenotransplantation. The experimental protocol was approved by the Kanazawa University Animal Care and Use Committee and conformed to the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences.