Engraftment of human mesenchymal stem cells in a severely immunodeficient mouse

Abstract

The transplantation of human mesenchymal stromal/stem cells (hMSCs) has potential as a curative and permanent therapy for congenital skeletal diseases. However, the self-renewal and differentiation capacities of hMSCs markedly vary. Therefore, cell proliferation and trilineage differentiation capacities were tested in vitro to characterize hMSCs before their clinical use. However, it remains unclear whether the ability of hMSCs in vitro accurately predicts that in living animals. The xenograft model is an alternative method for validating clinical MSCs. Nevertheless, the protocol still needs refinement, and it has yet to be established whether hMSCs, which are expanded in culture for clinical use, retain the ability to engraft and differentiate into adipogenic, osteogenic, and chondrogenic lineage cells in transplantation settings. In the present study, to establish a robust xenograft model of MSCs, we examined the delivery routes of hMSCs and the immunological state of recipients. The intra-arterial injection of hMSCs into X-ray-irradiated (IR) NOG, a severely immunodeficient mouse, achieved the highest engraftment but failed to sustain long-term engraftment. We demonstrated that graft cells localized to a collagenase-released fraction (CR), in which endogenous colony-forming cells reside. We also showed that Pdgfrα⁺Sca1⁺ MSCs (PαS), which reside in the CR fraction, resisted IR. These results show that our protocol enables hMSCs to fulfill a high level of engraftment in mouse bone marrow in the short term. In contrast, long-term reconstitution was restricted, at least partially, because of IR-resistant endogenous MSCs.

Keywords: Mesenchymal stem cells; Mesenchymal stromal cells; NOG; Regenerative medicine; Transplantation; Xenograft model.

Fig. 1

The CA injection improves MSC engraftment. A Experimental design. B One day after GFP + REC transplantation into C57BL6, CR (Left panel) or marrow (Right panel) fractions were prepared from a pair of tibia and femur and stained with the indicated antibodies for the FCM analysis. C Representative FCM profiles of the CR fraction from C57BL6 recipients transplanted with GFP + RECs through the CA or IV injection. D The frequencies of GFP + cells in the TN (Left panel) or stromal fraction (Right panel) were calculated from the FCM profile in (C). E Time course changes in GFP positivity in the TN or stromal fraction from C57BL6 transplanted with GFP-labeled REC (clones #3, 5, or 13) (E) or MSCs (F) are shown. Data are shown as the mean ± SEM. *P < .05; **P < .01; ***P < .005; ****P < .0005; *****P < .00005 by the Student’s t-test

Fig. 2

Immunosuppression improves MSC engraftment. A Frequencies of GFP⁺ cells in the TN (Left panel) or stromal fraction (Right panel) from mice transplanted with GFP-labeled RECs in the presence or absence of TAC on Day 7. B Experimental design. C GFP-labeled RECs were transplanted into NOG mice with or without semi-lethal IR through the CA or IV injection. Recipients were analyzed on Days 7 and 28. D The frequencies of GFP⁺ cells in the TN (Left panel) or CD90⁺ stromal fraction (Right panel) were calculated from the FCM profile in (C). E Number of GFP⁺ in a pair of tibia and femur. Data are shown as the mean ± SEM. *P < .05; **P < .01; ***P < .005; ****P < .0005; *****P < .00005 by the Student’s t-test

Fig. 3

Intra-bone marrow localization of RECs. Coimmunostaining of GFP and Osteocalcin (A-J) or GFP and Perilipin (K-Q) on the femur sections from the recipient seven days after transplantation. A, K GFP, (B) Osteocalcin, (C, M) DAPI, (D, N) Merged images, (L) Perilipin. E-J, O-Q Magnification images. Arrowheads indicate the GFP-expressing cells. Scale bar, 50 mm; CB, cortical bone

Fig. 4

PαS cells show IR resistance. C57BL6 mice were X-ray irradiated and the frequencies of PαS and CAR/LepR⁺ MSCs were examined on Days 0, 3, 7, and 10. A, B Representative FCM profiles of CAR/LepR⁺ MSCs (A) and the cellularity of the marrow fraction, frequencies, and absolute number of CAR/LepR⁺ MSCs (B) are shown. C, D Representative FCM profiles of PαS cells (C) and the cellularity of the CR fraction, frequencies, and absolute number of PαS cells (B) are shown. Data are shown as the mean ± SEM. *P < .05; **P < .01; ***P < .005; ****P < .0005; *****P < .00005 by the Student’s t-test