Researchers from the University of Minnesota Use Genetically Corrected Stem Cells To Repair Muscles


University of Minnesota researchers from the Lillehei Heart Institute have combined genetic engineering techniques to repair mutations in abnormal muscle cells with cellular reprogramming to generate stem cells that can repair and regenerate muscle regeneration in a mouse model for Duchenne Muscular Dystrophy (DMD). This research is a proof-of-principle experiment that determines the feasibility of combining induced pluripotent stem cell technology and genetic engineering techniques that correct mutations to treat muscular dystrophy. Experimental strategies such as these could represent a major step forward in autologous cell-based therapies for DMD. Furthermore, it might pave the way for clinical trials to test this approach in reprogrammed human pluripotent cells from muscular dystrophy patients.

University of Minnesota researchers combined three groundbreaking technologies to achieve effective muscular dystrophy therapy in a mouse model of DMD. First, researchers reprogrammed skin cells into induced pluripotent stem cells (iPSCs). iPSCs are capable of differentiating into any of the mature cell types within an adult organism. In this case, the University of Minnesota researchers generated pluripotent cells from the skin of mice that carry mutations in two genes; the dystrophin and utrophin genes. Mice with mutations in both the dystrophin and utrophin genes develop a severe case of muscular dystrophy that resembles the type of disease observed in human DMD patients. This provided a model system platform that successfully mimicked what would theoretically occur in humans.

The second technology employed is a genetic correction tool developed at the University of Minnesota. In this case, they used a transposon, which is a segment of DNA that can jump from one location to another within the genome. The specific transposon used is the “Sleeping Beauty Transposon.” The use of this transposon allowed them to transport genes into cells in a convenient manner. The Lillehei Heart Institute researchers used the Sleeping Beauty transposon to deliver a gene called “micro-utrophin” into the iPSCs made from the DMD mice.

Sleeping Beauty Transposon

Human micro-utrophin can support muscle fiber strength and prevent muscle fiber injury throughout the body. However, there is one essential difference micro-utrophin and dystrophin: dystrophin is absent in muscular dystrophy patients, but if it is introduced into the bodies of DMD patients, their immune system will initiate a devastating immune response against it. However, in those same patients, utrophin is active and functional, which makes it essentially “invisible” to the immune system. This invisibility allows the micro-utrophin to replace dystrophin build and repair muscle fibers within the body.

Utrophin

The third technology utilized is a method to produce skeletal muscle stem cells from pluripotent cells. This procedure was developed in the laboratory of Rita Perlingeiro, who is also the principal investigator of this latest study.

Rita Perlingeiro Ph.D.
Rita Perlingeiro Ph.D.

Perlingeiro’s technology gives pluripotent cells a short pulse of a muscle stem cell protein called Pax3, which nudges the pluripotent cells to become skeletal muscle stem cells, which can then be exponentially expanded in culture. These Pax3-induced muscle stem cells were then transplanted back into the same strain of DMD mice from which the pluripotent stem cells were originally derived.

Pax3 and 7

When combined, these platforms created muscle-generating stem cells that would not be rejected by the body’s immune system. According to Perlingeiro, the transplanted cells performed very well in the dystrophic mice, and they generated functional muscle and responded to muscle fiber injury.

“We were pleased to find the newly formed myofibers expressed the markers of the correction, including utrophin,” said Perlingeiro, a Lillehei endowed scholar within the Lillehei Heart Institute and an associate professor in the University of Minnesota Medical School. “However, a very important question following transplantation is if these corrected cells would self-renew, and produce new muscle stem cells in addition to the new muscle fibers.”

By injuring the transplanted muscle and watching it repair itself, the researchers demonstrated that the transplanted muscle stem cells endowed the recipient mice with fully functional muscle cells. This latest project provides the proof-of-principle for the feasibility of combining induced pluripotent stem cell technology and genetic correction to treat muscular dystrophy.

“Utilizing corrected induced pluripotent stem cells to target this specific genetic disease proved effective in restoring function,” said Antonio Filareto, Ph.D., a postdoctoral fellow in Perlingeiro’s laboratory and the lead author on the study. “These are very exciting times for research on muscular dystrophy therapies.”

These studies pave the way for testing this approach in a clinical trial that would use reprogrammed human pluripotent cells from muscular dystrophy patients.

According to Perlingeiro, “Developing methods to genetically repair muscular dystrophy in human cells, and demonstrating efficacy of muscle derived from these cells are critical near-term milestones, both for the field and for our laboratory. Testing in animal models is essential to developing effective technologies, but we remained focused on bringing these technologies into use in human cells and setting the stage for trials in human patients.”

This research was published in Nature Communications.

A Gene that Prevents Induced Pluripotent Stem Cell Formation Linked to Cancer Severity


A Mount Sinai research team has published some remarkable observations in the journal Nature Communications. Emily Bernstein, PhD, and her team at Mount Sinai have discovered a particular protein that prevents normal cells from being reprogrammed into induced pluripotent stem cells (iPSCs). Since iPSCs resemble embryonic stem cells, these data might provide significant insights into how cells lose their plasticity during normal development, which has wide-reaching implications for how cells change during both normal and disease development.

Previously, Bernstein and others showed that during the formation of particular tumors known as melanomas in mice and human patients, the loss of a specific histone variant called macroH2A (a protein that helps package DNA) correlated rather strongly to the growth and metastasis of the tumor. In this current study, Bernstein and her team determined if macroH2A acted as a barrier to cellular reprogramming during the derivation of iPSCs (see Costanzi C, Pehrson JR (1998). “Histone macroH2A1 is concentrated in the inactive X chromosome of female mammals”. Nature 393 (6685): 599–601).

In collaboration with researchers at the University of Pennsylvania, Bernstein evaluated mice that had been genetically engineered to lack macroH2A. When skin cells were used from macroH2A(-) mice were used to make iPSCs and compared with skin cells from macroH2A(+) mice, the cells from macroH2A(-) mice that lacked macroH2A were much more plastic and were much more easily reprogrammed into iPSCs compared to the wild-type or macroH2A(+) mice. This indicates that macroH2A may block cellular reprogramming by silencing genes required for plasticity.

Bernstein, who is an Assistant Professor of Oncological Sciences and Dermatology at the Graduate School of Biomedical Sciences at Mount Sinai, and corresponding author of the study, said: “This is the first evidence of the involvement of a histone variant protein as an epigenetic barrier to induced pluripotency (iPS) reprogramming.” She continued: “These findings help us to understand the progression of different cancers and how macroH2A might be acting as a barrier to tumor development.”

In their next group of experiments, Bernstein and her team plan to create cancer cells in a culture dish by inducing mutations in genes that are commonly abnormal in particular types of cancer cells and then couple those mutations to the removal of macroH2A to examine whether the cells are capable of forming tumors.