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Isolation of human foetal myoblasts and its application for microencapsulation.

Li AA, Bourgeois J, Potter M, Chang PL - J. Cell. Mol. Med. (2008 Jan-Feb)

Bottom Line: Foetal cells secrete more growth factors, generate less immune response, grow and proliferate better than adult cells.The lifespan of primary myoblasts was 70 population doublings before entering into senescent state, with a population time of 18-24 hrs.Hence, we have developed a protocol for isolating human foetal primary myoblasts with excellent differentiation potential and robust growth and longevity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology & Molecular Medicine, McMaster University, Hamilton, Ontario, Canada.

ABSTRACT
Foetal cells secrete more growth factors, generate less immune response, grow and proliferate better than adult cells. These characteristics make them desirable for recombinant modification and use in microencapsulated cellular gene therapeutics. We have established a system in vitro to obtain a pure population of primary human foetal myoblasts under several rounds of selection with non-collagen coated plates and identified by desmin staining. These primary myoblasts presented good proliferation ability and better differentiation characteristics in monolayer and after microencapsulation compared to murine myoblast C2C12 cells based on creatine phosphokinase (CPK), major histocompatibility complex (MHC) and multi-nucleated myotubule determination. The lifespan of primary myoblasts was 70 population doublings before entering into senescent state, with a population time of 18-24 hrs. Hence, we have developed a protocol for isolating human foetal primary myoblasts with excellent differentiation potential and robust growth and longevity. They should be useful for cell-based therapy in human clinical applications with microencapsulation technology.

Show MeSH
Creatine phosphokinase (CPK) activity in differentiation medium. The cells were cultured in growth medium for 24 hrs and switched into differentiation medium. The cells were trypsinized and pelleted by centrifugation at different time points. The pellets were suspended in phosphate buffered saline (PBS) and sonicated to release the cytoplasm. The CPK was assayed and standardized by protein concentration.
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fig02: Creatine phosphokinase (CPK) activity in differentiation medium. The cells were cultured in growth medium for 24 hrs and switched into differentiation medium. The cells were trypsinized and pelleted by centrifugation at different time points. The pellets were suspended in phosphate buffered saline (PBS) and sonicated to release the cytoplasm. The CPK was assayed and standardized by protein concentration.

Mentions: The differentiation potential of the primary myoblasts was assessed by CPK activity. Increased activity is characteristic of the onset of differentiation after changing from growth medium to a differentiation-inducing medium. As shown in Figure 2, over a period of 9 days in differentiation medium, the primary fibroblasts maintained only a baseline level of CPK activity. However, both primary myoblasts and C2C12 cells showed initially escalating levels that eventually decreased back to the baseline. While both cultures showed elevated CPK levels compared to the fibrob-lasts, the human myoblasts activity peaked early at day 3 whereas the C2C12 myoblasts showed the highest plateau from days 4–6. Furthermore, the specific CPK activity in the human myoblasts was greater than twofold higher than that achieved by the C2C12 myoblasts. The difference in the pattern of CPK activity indicated that the primary myoblasts differentiated earlier and more acutely than the C2C12.


Isolation of human foetal myoblasts and its application for microencapsulation.

Li AA, Bourgeois J, Potter M, Chang PL - J. Cell. Mol. Med. (2008 Jan-Feb)

Creatine phosphokinase (CPK) activity in differentiation medium. The cells were cultured in growth medium for 24 hrs and switched into differentiation medium. The cells were trypsinized and pelleted by centrifugation at different time points. The pellets were suspended in phosphate buffered saline (PBS) and sonicated to release the cytoplasm. The CPK was assayed and standardized by protein concentration.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3823488&req=5

fig02: Creatine phosphokinase (CPK) activity in differentiation medium. The cells were cultured in growth medium for 24 hrs and switched into differentiation medium. The cells were trypsinized and pelleted by centrifugation at different time points. The pellets were suspended in phosphate buffered saline (PBS) and sonicated to release the cytoplasm. The CPK was assayed and standardized by protein concentration.
Mentions: The differentiation potential of the primary myoblasts was assessed by CPK activity. Increased activity is characteristic of the onset of differentiation after changing from growth medium to a differentiation-inducing medium. As shown in Figure 2, over a period of 9 days in differentiation medium, the primary fibroblasts maintained only a baseline level of CPK activity. However, both primary myoblasts and C2C12 cells showed initially escalating levels that eventually decreased back to the baseline. While both cultures showed elevated CPK levels compared to the fibrob-lasts, the human myoblasts activity peaked early at day 3 whereas the C2C12 myoblasts showed the highest plateau from days 4–6. Furthermore, the specific CPK activity in the human myoblasts was greater than twofold higher than that achieved by the C2C12 myoblasts. The difference in the pattern of CPK activity indicated that the primary myoblasts differentiated earlier and more acutely than the C2C12.

Bottom Line: Foetal cells secrete more growth factors, generate less immune response, grow and proliferate better than adult cells.The lifespan of primary myoblasts was 70 population doublings before entering into senescent state, with a population time of 18-24 hrs.Hence, we have developed a protocol for isolating human foetal primary myoblasts with excellent differentiation potential and robust growth and longevity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology & Molecular Medicine, McMaster University, Hamilton, Ontario, Canada.

ABSTRACT
Foetal cells secrete more growth factors, generate less immune response, grow and proliferate better than adult cells. These characteristics make them desirable for recombinant modification and use in microencapsulated cellular gene therapeutics. We have established a system in vitro to obtain a pure population of primary human foetal myoblasts under several rounds of selection with non-collagen coated plates and identified by desmin staining. These primary myoblasts presented good proliferation ability and better differentiation characteristics in monolayer and after microencapsulation compared to murine myoblast C2C12 cells based on creatine phosphokinase (CPK), major histocompatibility complex (MHC) and multi-nucleated myotubule determination. The lifespan of primary myoblasts was 70 population doublings before entering into senescent state, with a population time of 18-24 hrs. Hence, we have developed a protocol for isolating human foetal primary myoblasts with excellent differentiation potential and robust growth and longevity. They should be useful for cell-based therapy in human clinical applications with microencapsulation technology.

Show MeSH