5 research outputs found
NANOG restores the impaired myogenic differentiation potential of skeletal myoblasts after multiple population doublings
Adult skeletal muscle regeneration relies on the activity of satellite cells residing in the skeletal muscle niche. However, systemic and intrinsic factors decrease the myogenic differentiation potential of satellite cells thereby impairing muscle regeneration. Here we present data showing that late passage C2C12 myoblasts exhibited significantly impaired myogenic differentiation potential that was accompanied by impaired expression of myogenic regulatory factors (Myf5, MyoD, Myogenin, and MRF4) and members of myocyte enhancer factor 2 family. Notably, ectopic expression of NANOG preserved the morphology and restored the myogenic differentiation capacity of late passage myoblasts, possibly by restoring the expression level of these myogenic factors. Muscle regeneration was effective in 2D cultures and in 3D skeletal microtissues mimicking the skeletal muscle niche. The presence of NANOG was required for at least 15 days to reverse the impaired differentiation potential of myoblasts. However, it was critical to remove NANOG during the process of maturation, as it inhibited myotube formation. Finally, myoblasts that were primed by NANOG maintained their differentiation capacity for 20 days after NANOG withdrawal, suggesting potential epigenetic changes. In conclusion, these results shed light on the potential of NANOG to restore the myogenic differentiation potential of myoblasts, which is impaired after multiple rounds of cellular division, and to reverse the loss of muscle regeneration. Keywords: Aging, Skeletal muscle loss, Sarcopenia, Satellite cells, C2C12 myoblasts, Myogenic differentiatio
Efficient and high yield isolation of myoblasts from skeletal muscle
Skeletal muscle (SkM) regeneration relies on the activity of myogenic progenitors that reside beneath the basal lamina of myofibers. Here, we describe a protocol for the isolation of the SkM progenitors from young and old mice by exploiting their outgrowth potential from SkM explants on matrigel coated dishes in the presence of high serum, chicken embryo extract and basic fibroblast growth factor. Compared to other protocols, this method yields a higher number of myoblasts (10–20 million) by enabling the outgrowth of these cells from tissue fragments. The majority of outgrowth cells (~90%) were positive for myogenic markers such as α7-integrin, MyoD, and Desmin. The myogenic cell population could be purified to 98% with one round of pre-plating on collagen coated dishes, where differential attachment of fibroblasts and other non-myogenic progenitors separates them from myoblasts. Moreover, the combination of high serum medium and matrigel coating provided a proliferation advantage to myogenic cells, which expanded rapidly (~24 h population doubling), while non-myogenic cells diminished over time, thereby eliminating the need for further purification steps such as FACS sorting. Finally, myogenic progenitors gave rise to multinucleated myotubes that exhibited sarcomeres and spontaneous beating in the culture dish. Keywords: Skeletal muscle progenitors, Myoblast isolation, Satellite cells, Myogenic differentiatio
Well-Defined pH-Responsive Self-Assembled Block Copolymers for the Effective Codelivery of Doxorubicin and Antisense Oligonucleotide to Breast Cancer Cells
The worldwide steady increase in the number of cancer
patients
motivates the development of innovative drug delivery systems for
combination therapy as an effective clinical modality for cancer treatment.
Here, we explored a design concept based on poly(ethylene glycol)-b-poly(2-(dimethylamino)ethyl methacrylate)-b-poly(2-hydroxyethyl methacrylate-formylbenzoic acid) [PEG-b-PDMAEMA-b-P(HEMA-FBA)] for the dual delivery
of doxorubicin (DOX) and GTI2040 (an antisense oligonucleotide for
ribonucleotide reductase inhibition) to MCF-7 breast cancer cells.
PEG-b-PDMAEMA-b-PHEMA, the precursor
copolymer, was prepared through chain extensions from a PEG-based
macroinitiator via two consecutive atom transfer radical polymerization
(ATRP) steps. Then, it was modified at the PHEMA block with 4-formylbenzoic
acid (FBA) to install reactive aldehyde moieties. A pH-responsive
polymer–drug conjugate (PDC) was obtained by conjugating DOX
to the polymer structure via acid-labile imine linkages, and subsequently
self-assembled in an aqueous solution to form DOX-loaded self-assembled
nanoparticles (DOX-SAN) with a positively charged shell. DOX-SAN condensed
readily with negatively charged GTI2040 to form GTI2040/DOX-SAN nanocomplexes.
Gel-retardation assay confirmed the affinity between GTI2040 and DOX-SAN.
The GTI2040/DOX-SAN nanocomplex at N/P ratio of 30 exhibited a volume-average
hydrodynamic size of 136.4 nm and a zeta potential of 21.0 mV. The
pH-sensitivity of DOX-SAN was confirmed by the DOX release study based
on the significant cumulative DOX release at pH 5.5 relative to pH
7.4. Cellular uptake study demonstrated favorable accumulation of
GTI2040/DOX-SAN inside MCF-7 cells compared with free GTI2040/DOX.
In vitro cytotoxicity study indicated higher therapeutic efficacy
of GTI2040/DOX-SAN relative to DOX-SAN alone because of the downregulation
of the R2 protein of ribonucleotide reductase. These outcomes suggest
that the self-assembled pH-responsive triblock copolymer is a promising
platform for combination therapy, which may be more effective in combating
cancer than individual therapies