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, 112 (2), 530-5

MSCs Derived From iPSCs With a Modified Protocol Are Tumor-Tropic but Have Much Less Potential to Promote Tumors Than Bone Marrow MSCs

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MSCs Derived From iPSCs With a Modified Protocol Are Tumor-Tropic but Have Much Less Potential to Promote Tumors Than Bone Marrow MSCs

Qingguo Zhao et al. Proc Natl Acad Sci U S A.

Abstract

Mesenchymal stem or stromal cells (MSCs) have many potential therapeutic applications including therapies for cancers and tissue damages caused by cancers or radical cancer treatments. However, tissue-derived MSCs such as bone marrow MSCs (BM-MSCs) may promote cancer progression and have considerable donor variations and limited expandability. These issues hinder the potential applications of MSCs, especially those in cancer patients. To circumvent these issues, we derived MSCs from transgene-free human induced pluripotent stem cells (iPSCs) efficiently with a modified protocol that eliminated the need of flow cytometric sorting. Our iPSC-derived MSCs were readily expandable, but still underwent senescence after prolonged culture and did not form teratomas. These iPSC-derived MSCs homed to cancers with efficiencies similar to BM-MSCs but were much less prone than BM-MSCs to promote the epithelial-mesenchymal transition, invasion, stemness, and growth of cancer cells. The observations were probably explained by the much lower expression of receptors for interleukin-1 and TGFβ, downstream protumor factors, and hyaluronan and its cofactor TSG6, which all contribute to the protumor effects of BM-MSCs. The data suggest that iPSC-derived MSCs prepared with the modified protocol are a safer and better alternative to BM-MSCs for therapeutic applications in cancer patients. The protocol is scalable and can be used to prepare the large number of cells required for "off-the-shelf" therapies and bioengineering applications.

Keywords: cancer; iPS cells; mesenchymal stem cells; protumor effects; tumor tropism.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Characterization of iPSC-MSCs. (A) Derivation and morphology of MSC-like cells from human iPSCs. (B) qRT-PCR analysis of relative expression of marker genes for pluripotency and each germ layer in iPSCs, BM-MSCs, and iPSC-MSCs (**P < 0.01 vs. iPSCs; ND, not detected). (C) Flow cytometry analysis of surface markers in iPSC-MSCs. (D and E) Multilineage differentiation of iPSC-MSCs. After 2 wk of corresponding induction, differentiated iPSC-MSCs were stained for mineralization with Alizarin Red, for chondrocytes with Toluidine Blue, or for lipid drops with Oil Red, respectively (D), and analyzed for expression of marker genes of osteoblasts, chondrocytes, or adipocytes, respectively, by qRT-PCR in comparison with identically differentiated BM-MSCs (E). (F) Telomerase activities in iPSCs, BM-MSCs, and iPSC-MSCs. (G) CFU–F-forming assay. *P < 0.05. (H) Growth curves of BM-MSCs and iPSC-MSCs (n = 3). iPSC-MSCs ceased expanding after 17 passages (64 population doublings) and BM-MSCs after 16 passages (48 population doublings).
Fig. 2.
Fig. 2.
The tumor tropism of iPSC-MSCs. (A) qRT-PCR analysis of genes related to tumor homing in MSCs. (B) In vitro migration of MSCs toward 293T or MDA-MB-231 cells in transwells. (C) Standard curve for qPCR assays of MSCs carrying CMV-copGFP added into LoVo or MDA-MB-231 cancer xenografts. Values indicate ∆Ct for primers for CMV promoter and mouse/human GAPDH genes on same samples; n = 3. (D and E) Estimated percentage of homed MSCs carrying CMV-copGFP in all tumor cells in the LoVo or MDA-MB-231 cancer xenograft model based on qPCR of CMV promoter; n = 5. (F) Homing of i.v. infused GFP-MSCs to established s.c. LoVo or MDA-MB-231 cancer xenografts was confirmed by immunofluorescence staining of copGFP in frozen sections.
Fig. 3.
Fig. 3.
The effects of iPSC-MSC on EMT, invasion, and cancer stem cells of cocultured cancer cells. (AD) After coculture with GFP-labeled BM-MSCs or iPS-MSCs, cancer cells were isolated by FACS and subjected to (A) qRT-PCR analysis of genes related to EMT and invasion. (B) Invasion assay with collagen IV-coated Boyden chambers. (C) Representative flow cytometry analysis of ALDH+ population and the percentage of ALDH+ cells (mean ± SEM of three independent tests). (D) Mammosphere-forming assay of breast cancer cells. (E) Weights of tumors derived from HCC1806 cells injected into SCID mice with BM- or iPSC-MSCs at 6 wk.
Fig. 4.
Fig. 4.
ILR-PGE2-IL6 pathway in iPSC-MSCs. (A) qRT-PCR analysis of genes of the ILR-PGE2-IL6 pathway in MSCs cultured with αMEM, IL1, LoVo conditioned medium (TCM), or cells for 3 d. (B) ELISA of PGE2 in 3-d medium of MSCs cultured under above conditions.
Fig. 5.
Fig. 5.
TGFβ-SDF1 pathway in iPSC-MSCs. (A) qRT-PCR analysis of TGFβ receptors (TGFBR1 and TGFBR2) and TGFβ target genes (ID3, IL6, PAI1, and SDF1) in MSCs cultured with αMEM, TGFβ, or MDA-MB-231–conditioned medium (TCM) for 3 d. (B) Western blot analysis of levels of phospho-Smad2 and phospho-Smad3 in MSCs cultured under above conditions, normalized to GAPDH (n = 3).
Fig. 6.
Fig. 6.
The expression of HASs and TSG6 and the HA production in MSCs and the induction of LOX in cancer cells. (A) qRT-PCR analysis of HAS1-3 and TSG6 in MSCs cultured alone or with LoVo cells for 3 d. (B) Enzyme immunoassay (EIA) analysis of HA in 6-d medium of BM-MSCs or iPSC-MSCs at passages 5 and 15. (C) qRT-PCR analysis of LOX in cancer cells cultured alone or with MSCs for 3 d.

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