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Bovine dedifferentiated adipose tissue (DFAT) cells: DFAT cell isolation.

Wei S, Du M, Jiang Z, Duarte MS, Fernyhough-Culver M, Albrecht E, Will K, Zan L, Hausman GJ, Elabd EM, Bergen WG, Basu U, Dodson MV - Adipocyte (2013)

Bottom Line: One to 30% DFAT cells redifferentiated into lipid-assimilating adipocytes in the DMI media, with distinct lipid-droplets in the cytoplasm and with no observable lipid-free vesicles inside.Moreover, a high confluence level promoted the redifferentiation efficiency of DFAT cells.Wagyu IMF dedifferentiated DFAT cells exhibited unique adipogenesis modes in vitro, revealing a useful cell model for studying adipogenesis and lipid metabolism.

View Article: PubMed Central - PubMed

Affiliation: College of Animal Science and Technology; Northwest A&F University; Yangling, Shaanxi Province PR China ; Department of Animal Sciences; Washington State University; Pullman, WA USA.

ABSTRACT
Dedifferentiated fat cells (DFAT cells) are derived from lipid-containing (mature) adipocytes, which possess the ability to symmetrically or asymmetrically proliferate, replicate, and redifferentiate/transdifferentiate. Robust cell isolation and downstream culture methods are needed to isolate large numbers of DFAT cells from any (one) adipose depot in order to establish population dynamics and regulation of the cells within and across laboratories. In order to establish more consistent/repeatable methodology here we report on two different methods to establish viable DFAT cell cultures: both traditional cell culture flasks and non-traditional (flat) cell culture plates were used for ceiling culture establishment. Adipocytes (maternal cells of the DFAT cells) were easier to remove from flat culture plates than flasks and the flat plates also allowed cloning rings to be utilized for cell/cell population isolation. While additional aspects of usage of flat-bottomed cell culture plates may yet need to be optimized by definition of optimum bio-coating to enhance cell attachment, utilization of flat plate approaches will allow more efficient study of the dedifferentiation process or the DFAT progeny cells. To extend our preliminary observations, dedifferentiation of Wagyu intramuscular fat (IMF)-derived mature adipocytes and redifferentiation ability of DFAT cells utilizing the aforementioned isolation protocols were examined in traditional basal media/differentiation induction media (DMI) containing adipogenic inducement reagents. In the absence of treatment approximately 10% isolated Wagyu IMF-mature adipocytes dedifferentiated spontaneously and 70% DFAT cells displayed protracted adipogenesis 12 d after confluence in vitro. Lipid-free intracellular vesicles in the cytoplasm (vesicles possessing an intact membrane but with no any observable or stainable lipid inside) were observed during redifferentiation. One to 30% DFAT cells redifferentiated into lipid-assimilating adipocytes in the DMI media, with distinct lipid-droplets in the cytoplasm and with no observable lipid-free vesicles inside. Moreover, a high confluence level promoted the redifferentiation efficiency of DFAT cells. Wagyu IMF dedifferentiated DFAT cells exhibited unique adipogenesis modes in vitro, revealing a useful cell model for studying adipogenesis and lipid metabolism.

No MeSH data available.


Related in: MedlinePlus

Figure 5. Transmission electron microscopy of lipid-free vesicles in DFAT cells. Lipid-free vesicles were intact and surrounded by a phospholipid monolayer. [Scale bars: (A) 0.5 µm; (B) 0.2 µm]
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Figure 5: Figure 5. Transmission electron microscopy of lipid-free vesicles in DFAT cells. Lipid-free vesicles were intact and surrounded by a phospholipid monolayer. [Scale bars: (A) 0.5 µm; (B) 0.2 µm]

Mentions: In the present study, DFAT cells (Fig. 4A) redifferentiated into adipocytes 10 d after applying the FBS-based DMI media. These adipocytes acquired cytoplasmic lipid droplets that were visible by the contrast microscope (Fig. 4B and D) and were confirmed by ORO staining (Fig. 4C and E). Although cells of a single DFAT clone were derived from one mother cell (one lipid-filled mature adipocyte), potential adipogenesis abilities of these cells differed. Few cells in 20% and 50% confluence groups accumulated lipids, cells in the 80% confluence group displayed less than 10% differentiation (Fig. 4E) and DFAT cells in the 100% confluence group had the highest rate of differentiation (around 30%) (Fig. 4C), demonstrating that increased seeding density as well as increased confluence prior to induction by DMI media may promote DFAT cell redifferentiation. Comparing with 100% confluence group cultures exposed to DMI, the control cultures (no application of inducement components) grew into numerous foci (cells grew on top of one another after achieving the maximal cell numbers spread in cell culture plate)41 and lipid devoid intracellular vesicles appeared after 12 d of confluence, which was confirmed by ORO staining (Fig. 4F). In addition, The TEM performed on the vesicles in cells of control cultures illustrated that the vesicles were intact and surrounded by a phospholipid monolayer, indicating the lipid-free vesicles were capable of packaging and holding lipids (Fig. 5). These results support the theory that DFAT cells could differentiate into “immature adipocyte-like” cells,41 with cytoplasmic lipid devoid, but membrane-intact, vesicles.


Bovine dedifferentiated adipose tissue (DFAT) cells: DFAT cell isolation.

Wei S, Du M, Jiang Z, Duarte MS, Fernyhough-Culver M, Albrecht E, Will K, Zan L, Hausman GJ, Elabd EM, Bergen WG, Basu U, Dodson MV - Adipocyte (2013)

Figure 5. Transmission electron microscopy of lipid-free vesicles in DFAT cells. Lipid-free vesicles were intact and surrounded by a phospholipid monolayer. [Scale bars: (A) 0.5 µm; (B) 0.2 µm]
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3756103&req=5

Figure 5: Figure 5. Transmission electron microscopy of lipid-free vesicles in DFAT cells. Lipid-free vesicles were intact and surrounded by a phospholipid monolayer. [Scale bars: (A) 0.5 µm; (B) 0.2 µm]
Mentions: In the present study, DFAT cells (Fig. 4A) redifferentiated into adipocytes 10 d after applying the FBS-based DMI media. These adipocytes acquired cytoplasmic lipid droplets that were visible by the contrast microscope (Fig. 4B and D) and were confirmed by ORO staining (Fig. 4C and E). Although cells of a single DFAT clone were derived from one mother cell (one lipid-filled mature adipocyte), potential adipogenesis abilities of these cells differed. Few cells in 20% and 50% confluence groups accumulated lipids, cells in the 80% confluence group displayed less than 10% differentiation (Fig. 4E) and DFAT cells in the 100% confluence group had the highest rate of differentiation (around 30%) (Fig. 4C), demonstrating that increased seeding density as well as increased confluence prior to induction by DMI media may promote DFAT cell redifferentiation. Comparing with 100% confluence group cultures exposed to DMI, the control cultures (no application of inducement components) grew into numerous foci (cells grew on top of one another after achieving the maximal cell numbers spread in cell culture plate)41 and lipid devoid intracellular vesicles appeared after 12 d of confluence, which was confirmed by ORO staining (Fig. 4F). In addition, The TEM performed on the vesicles in cells of control cultures illustrated that the vesicles were intact and surrounded by a phospholipid monolayer, indicating the lipid-free vesicles were capable of packaging and holding lipids (Fig. 5). These results support the theory that DFAT cells could differentiate into “immature adipocyte-like” cells,41 with cytoplasmic lipid devoid, but membrane-intact, vesicles.

Bottom Line: One to 30% DFAT cells redifferentiated into lipid-assimilating adipocytes in the DMI media, with distinct lipid-droplets in the cytoplasm and with no observable lipid-free vesicles inside.Moreover, a high confluence level promoted the redifferentiation efficiency of DFAT cells.Wagyu IMF dedifferentiated DFAT cells exhibited unique adipogenesis modes in vitro, revealing a useful cell model for studying adipogenesis and lipid metabolism.

View Article: PubMed Central - PubMed

Affiliation: College of Animal Science and Technology; Northwest A&F University; Yangling, Shaanxi Province PR China ; Department of Animal Sciences; Washington State University; Pullman, WA USA.

ABSTRACT
Dedifferentiated fat cells (DFAT cells) are derived from lipid-containing (mature) adipocytes, which possess the ability to symmetrically or asymmetrically proliferate, replicate, and redifferentiate/transdifferentiate. Robust cell isolation and downstream culture methods are needed to isolate large numbers of DFAT cells from any (one) adipose depot in order to establish population dynamics and regulation of the cells within and across laboratories. In order to establish more consistent/repeatable methodology here we report on two different methods to establish viable DFAT cell cultures: both traditional cell culture flasks and non-traditional (flat) cell culture plates were used for ceiling culture establishment. Adipocytes (maternal cells of the DFAT cells) were easier to remove from flat culture plates than flasks and the flat plates also allowed cloning rings to be utilized for cell/cell population isolation. While additional aspects of usage of flat-bottomed cell culture plates may yet need to be optimized by definition of optimum bio-coating to enhance cell attachment, utilization of flat plate approaches will allow more efficient study of the dedifferentiation process or the DFAT progeny cells. To extend our preliminary observations, dedifferentiation of Wagyu intramuscular fat (IMF)-derived mature adipocytes and redifferentiation ability of DFAT cells utilizing the aforementioned isolation protocols were examined in traditional basal media/differentiation induction media (DMI) containing adipogenic inducement reagents. In the absence of treatment approximately 10% isolated Wagyu IMF-mature adipocytes dedifferentiated spontaneously and 70% DFAT cells displayed protracted adipogenesis 12 d after confluence in vitro. Lipid-free intracellular vesicles in the cytoplasm (vesicles possessing an intact membrane but with no any observable or stainable lipid inside) were observed during redifferentiation. One to 30% DFAT cells redifferentiated into lipid-assimilating adipocytes in the DMI media, with distinct lipid-droplets in the cytoplasm and with no observable lipid-free vesicles inside. Moreover, a high confluence level promoted the redifferentiation efficiency of DFAT cells. Wagyu IMF dedifferentiated DFAT cells exhibited unique adipogenesis modes in vitro, revealing a useful cell model for studying adipogenesis and lipid metabolism.

No MeSH data available.


Related in: MedlinePlus