Characteristics of Ad-MSCs, BM-MSCs, and XF-iMSCs. Credit: Stem Cell Research & Therapy (2024). DOI: 10.1186/s13287-024-03951-6

A research team led by Associate Professor Hidetoshi Sakurai and Researcher Nana Takenaka-Ninagawa recently demonstrated the superior therapeutic potential of iPS cell-derived mesenchymal stromal cells (iMSCs) compared to primary MSCs as a potential treatment for Ullrich congenital muscular dystrophy. The study is published in Stem Cell Research & Therapy.

Ullrich congenital muscular dystrophy (UCMD) is an early-onset, progressive muscular disease characterized by muscle weakness and joint contractures, ultimately leading to respiratory difficulties. It can be caused by mutations in any of the three genes encoding type 6 collagen, COL6A1, COL6A2, or COL6A3, that alter the extracellular matrix (ECM), a network of proteins and other molecules supporting and providing structure to cells and tissues in the body.

MSCs secrete type 6 collagen and help maintain homeostasis in skeletal muscle tissues, thus highlighting it as a potential therapeutic target for UCMD treatment. The Sakurai Laboratory previously demonstrated that iMSCs promote muscle regeneration and maturation upon administration to mice lacking the Col6a1 gene. However, MSCs can also be derived from various tissues, such as adipose tissue and bone marrow, some of which are already being clinically applied.

Nevertheless, no studies to date have compared or verified the therapeutic effects of these MSCs in UCMD model mice. To better understand their differences, as it will undoubtedly help to guide future developments of MSC-based cell therapies for UCMD and other diseases, the research team performed a comprehensive comparison between MSCs from different sources.

As a start, the researchers observed primary Ad-MSCs and BM-MSCs to show the same morphology and cell-type specific markers as iMSCs and, importantly, all expressed type 6 collagen at the RNA and protein levels. With this knowledge, they performed transplantation studies by injecting these different MSCs into the tibialis anterior muscle—commonly known as the shin muscle—of Col6a1 knockout (KO) mice.

As expected, all MSCs, regardless of source, grafted within a week of transplantation, with COL6 supplementation readily observable. Notably, BM-MSC transplantation resulted in the highest COL6-positive area and muscle fibers, with iMSCs and Ad-MSCs restoring COL6 to lower but comparable levels.

Whereas muscle fibers showed numerous signs of incomplete maturation in both the Ad-MSC- and BM-MSC-transplanted groups, iMSC-transplanted Col6a1 KO mice possessed the most multinucleated regenerated muscle fibers and larger fibers than other transplant groups. These observations continued up to 12 weeks following transplantation as Col6a1 KO mice transplanted with BM-MSCs once again displayed the most COL6-positive area and muscle fibers.

However, like observations from 1 week after transplantation, iMSC-transplanted animals showed the most muscle maturation, with few smaller, immature muscle fibers, comparable to normal healthy mice.

By contrast, Ad-MSC- and BM-MSC-transplanted Col6a1 KO animals exhibited no difference in the number of smaller muscle fibers compared to untreated Col6a1 KO mice. Thus, paradoxically, whereas iMSC transplantation resulted in the lowest number of cells at both 1 and 12 weeks after transplantation, they provided the most therapeutic benefit among MSCs tested, causing the team to hypothesize that Ad-MSCs and BM-MSCs may also have had detrimental effects on muscle fibers.

To this end, the researchers examined the level of fibrosis in muscles following transplantation. From this investigation, they observed significant fibrosis in mice transplanted with BM-MSCs. While Ad-MSC transplantation also led to some fibrosis, animals with iMSCs transplanted did not show any fibrosis or stromal expansion, even up to 24 weeks after transplantation.

To determine the underlying cause for the differences in the ability of different MSCs to exert therapeutic effects in Col6a1 KO mice, the researchers used a co-culture system consisting of muscle stem cells (MuSCs) from Col6a1 KO mice and the different MSCs.

Through this co-culture system, they observed that while co-culturing MuSCs with MSCs enhanced myogenic differentiation, iMSCs were the most effective and promoted the formation of more multinucleated, elongated muscle fibers, like in animal experiments.

Because other studies have previously shown proteins such as IGF2 and peroxidasin to enhance myogenic differentiation, the research team examined whether they were involved in the observations here.

Gene expression analysis determined that iMSCs expressed the IGF2 gene at much higher levels than other MSCs, and while the expression of the PXDN gene was moderately higher in iMSCs, the change compared to other MSCs was not statistically different. The researchers consistently detected higher levels of IGF2 protein produced by iMSCs than Ad- or BM-MSCs.

Remarkably, when the researchers knocked down IGF2 in iMSCs before co-culturing with MuSCs, their ability to promote myogenic differentiation was severely impaired, thus demonstrating the critical involvement of IGF2.

Finally, they tested whether IGF2 treatment of MuSCs without MSCs would show the same therapeutic benefits and similarly observed enhanced myogenic differentiation by simply treating MuSCs with IGF2.

From this work, the research team revealed that, compared to primary MSCs, iMSCs are superior in promoting myogenic differentiation in an IGF2-dependent manner. While it is clear from their work that additional factors produced by iMSCs or cell-cell interactions may be involved, this work gives hope that future studies will identify other crucial mechanisms that could form the basis of effective treatments for UCMD.