UCLA scientists have developed a new strategy to efficiently
isolate, mature and transplant skeletal muscle cells created from human
pluripotent stem cells, which can produce all cell types of the body. The
findings are a major step towards developing a stem cell replacement therapy
for muscle diseases including Duchenne Muscular Dystrophy, which affects
approximately 1 in 5,000 boys in the U.S. and is the most common fatal
childhood genetic disease.
The study was published in the journal Nature Cell Biology
by senior author April Pyle, associate professor of microbiology, immunology
and molecular genetics and member of the Eli and Edythe Broad Center of
Regenerative Medicine and Stem Cell Research at UCLA. Using the natural human
development process as a guide, the researchers developed ways to mature muscle
cells in the laboratory to create muscle fibers that restore dystrophin, the
protein that is missing in the muscles of boys with Duchenne…
For years, scientists have been trying different methods
that direct human pluripotent stem cells to generate skeletal muscle stem cells
that can function appropriately in living muscle and regenerate
dystrophin-producing muscle fibers. However, the study led by Pyle found that
the current methods are inefficient; they produce immature cells that are not
appropriate for modeling Duchenne in the laboratory or creating a cell
replacement therapy for the disease.
“We have found that just because a skeletal muscle cell
produced in the lab expresses muscle markers, doesn’t mean it is fully
functional,” said Pyle. “For a stem cell therapy for Duchenne to move forward,
we must have a better understanding of the cells we are generating from human
pluripotent stem cells compared to the muscle stem cells found naturally in the
human body and during the development process.”
By analyzing human development, the researchers found a
fetal skeletal muscle cell that is extraordinarily regenerative. Upon further
analysis of these fetal muscle cells two new cell surface markers called ERBB3
and NGFR were discovered; this enabled the reserchers to precisely isolate
muscle cells from human tissue and separate them from various cell types created
using human pluripotent stem cells.
Once they were able to isolate skeletal muscle cells using the newly
identified surface markers, the research team matured those cells in the lab to
create dystrophin-producing muscle fibers. The muscle fibers they created were
uniformily muscle cells, but the fibers were still smaller than those found in
real human muscle.
“We were missing another key component,” said Michael Hicks,
lead author of the study. The skeletal muscle cells were not maturing properly,
he explained. “We needed bigger, stronger muscle that also had the ability to
contract.”
Once again, the team looked to the natural stages of human
development for answers. Hicks discovered that a specific cell signaling
pathway called TGF Beta needs to be turned off to enable generation of skeletal
muscle fibers that contain the proteins that help muscles contract. Finally,
the team tested their new method in a mouse model of Duchenne.
“Our long term goal is to develop a personalized cell
replacement therapy using a patient’s own cells to treat boys with Duchenne,”
said Hicks. “So, for this study we followed the same steps, from start to
finish, that we’d follow when creating these cells for a human patient.”
First, the Duchenne patient cells were reprogrammed to
become pluripotent stem cells. The researchers then removed the genetic
mutation that causes Duchenne using the gene editing technology CRISPR-Cas9.
Using the ERBB3 and NGFR surface markers, the skeletal muscle cells were
isolated and then injected into mice at the same time a TGF Beta inhibitor was
administered.
“The results were exactly what we’d hoped for,” said Pyle.
“This is the first study to demonstrate that functional muscle cells can be
created in a laboratory and restore dystrophin in animal models of Duchenne
using the human development process as a guide.”
Courtesy of: https://neurologistconnect.com/posts/5a427355634e8877378b4578?SKUID=6656d46c04553656b04bf4a8e0248071&mkt_tok=eyJpIjoiTldNNVptSTBaakkzTURRNCIsInQiOiJjSkVES29IVzY3RmVBMHZvT0pBY2xTdUQ4NDJLT2Z6VmhpWTVHU3ZnNHpJRkJWZXZGNVYwcnlRdUlhenBCQklIR2Y2STRscVBwYjJROE5MTUJpOFlcL0R3eGx2THY3Wnl4K2s5RjRFM0F6cFpoaUNQNWZrMndvTXkxak43dERcLzhqIn0%3D
Hicks MR, Hiserodt J, Paras K, Fujiwara W, Eskin A, Jan M,
Xi H, Young CS, Evseenko D, Nelson SF, Spencer MJ, Handel BV, Pyle AD. ERBB3
and NGFR mark a distinct skeletal muscle progenitor cell in human
development and hPSCs. Nat Cell Biol. 2018 Jan;20(1):46-57.
Abstract
Human pluripotent stem cells (hPSCs) can be directed to
differentiate into skeletal muscle progenitor cells (SMPCs). However, the
myogenicity of hPSC-SMPCs relative to human fetal or adult satellite cells
remains unclear. We observed that hPSC-SMPCs derived by directed
differentiation are less functional in vitro and in vivo compared to human
satellite cells. Using RNA sequencing, we found that the cell surface receptors
ERBB3 and NGFR demarcate myogenic populations, including PAX7 progenitors in
human fetal development and hPSC-SMPCs. We demonstrated that hPSC skeletal
muscle is immature, but inhibition of transforming growth factor-β signalling
during differentiation improved fusion efficiency, ultrastructural organization
and the expression of adult myosins. This enrichment and maturation strategy
restored dystrophin in hundreds of dystrophin-deficient myofibres after
engraftment of CRISPR-Cas9-corrected Duchenne muscular dystrophy human induced
pluripotent stem cell-SMPCs. The work provides an in-depth characterization of
human myogenesis, and identifies candidates that improve the in vivo myogenic
potential of hPSC-SMPCs to levels that are equal to directly isolated human
fetal muscle cells.
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