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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 27, 2005 as doi:10.1096/fj.04-3204fje. |
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Pennington Biomedical Research Center, Baton Rouge, Louisiana, USA
1 Correspondence: Pennington Biomedical Research Center, 6400 Perkins Rd., Baton Rouge, LA 70808, USA. E-mail: kozakb{at}pbrc.edu
SPECIFIC AIMS
We have identified ear mesenchymal stem cells (EMSC) as a novel source of adult stem cells from the external ears of mice. The objectives of the present study were to 1) investigate the conditions for commitment of EMSC to the adipogenic lineage; 2) compare adipogenesis of EMSC to that of the stromal-vascular (S-V) cells present in fat depots of mice; and 3) generate and characterize clones of EMSC.
PRINCIPAL FINDINGS
1. Adipogenic potential of EMSC
Cells were collected from external ears of 3 different strains of mice (Hsd: Athymic Nude-nu, FVB, and C57BL/6J). At confluence, EMSC at each of 5 passages were stimulated to differentiate into adipocytes by adding differentiation medium (DMEM/F12 plus 4% FBS supplemented with 1.7 µM insulin, 1 µM dexamethasone, and 0.5 mM isobutyl-methylxanthine). After 5 days in differentiation medium, EMSC show an abundance of oil red O-stained lipid droplets. Virtually no staining was found in EMSC cultured in medium without differentiation factors. Multiple differentiation experiments with EMSC collected from the ears of different strains of mice showed no differences in adipogenic abilities among strains. The phenotypical changes during adipocyte differentiation were associated with the expression of adipocyte-specific genes. The differentiation of EMSC into adipocytes was verified by RT-PCR of PPAR
, aP2, and LPL in total RNA from control/unstimulated cultures and from the cultures stimulated by differentiation medium for 5 days. Culture from all 3 strains of mice showed robust adipocyte differentiation as far as passage 5.
Leptin secretion was determined in conditioned medium collected from EMSC cultures. Confluent EMSC were stimulated with differentiation medium for 3, 6, 12, 24, 48, 72, 96, and 120 h. After stimulation, cultures were changed to DMEM/F12 medium with 4% FBS and 10 nM insulin and maintained for up to 168 h. Control cultures without adipogenic stimulation were maintained in DMEM/F12 medium containing 4% FBS and 10 nM insulin. Leptin assays were performed using 48 h conditioned medium in the presence of 4% FBS. Level of leptin secretion increased from the 68 pg/mL (3 h adipogenic stimulation) to achieve a maximum level of 1483 pg/mL after 48 h.
Differentiated EMSC have shown increased glucose uptake and lipolysis in response to insulin and ß-adrenergic stimulation, respectively. After differentiation, EMSC demonstrated higher capacity and showed insulin-dependent glucose uptake. We examined the phosphorylation of AKT, a downstream kinase involved in insulin stimulated glucose uptake. Stimulation of AKT phosphorylation to a peak level is observed within 30 min in differentiated EMSC with a slight increase in GSK3ß phosphorylation associated with the stimulation of glycogen synthesis. Similarly, norepinephrine stimulates phosphorylation of CREB and p38 MAPK, targets for PKA activation, from differentiated EMSC. Our data showed no significant changes in RIIß phosphorylation by norepinephrine treatment, suggesting that RIIß might be a lesser contributor than RI
in EMSC.
2. Adipogenic differentiation of EMSC vs. preadipocytes from S-V fraction
To compare adipogenic properties of EMSC to preadipocytes from S-V fraction, ear and inguinal fat depot samples were collected from the same animals and isolated cells were cultured under the same conditions. Morphological changes in adipogenic differentiation were assessed by staining fixed cultures with oil red O (Fig. 1
A). Lipid accumulation was much denser in EMSC than S-V at the same time point of differentiation. Marked increase in lipid droplets was observed in EMSC after TZD treatment. To quantify accumulation of lipid, oil red O was extracted and absorbance was measured at 500 nm (Fig. 1B
). Whereas adipogenic differentiation of S-V cells from fat tissues was low, that from the EMSC was extremely robust. This experiment was repeated several times using EMSC and S-V cells from C57BL/6J and FVB strains; differentiation of EMSC was consistently robust, whereas that of S-V cells was highly variable, often very low (Fig. 1BI, II
). To quantify differentiation of EMSC vs. S-V cells into adipocytes, we measured expression of adipogenic markers, PPAR and aP2, using qRT-PCR. After 9 days in culture expression of aP2 mRNA increased 50-fold for EMSC and 8-fold for S-V cells compared with the control/unstimulated cultures. PPAR mRNA, as an early differentiation marker, showed significantly higher expression in EMSC at the third day of stimulation. TZD induced PPAR expression more dramatically in EMSC than S-V fraction. Changes in expression of adipogenic markers were confirmed by Western blot analysis. As in earlier studies, expression of PPAR gradually increased during EMSC differentiation. We observed PPAR1 and PPAR2 from differentiated EMSC. In this study protein level of C/EBP
gradually increased until 5 days of differentiation in parallel with PPAR2 and levels were stably maintained during the subsequent days of EMSC differentiation. This pattern of C/EBP expression in EMSC is different from its early transient expression in the early stages of adipogenesis in 3T3-L1 cells. Since lipolysis and thermogenesis from white and brown adipocytes are regulated by SNS activation through ß-adrenergic receptor, cAMP-mediated PKA and downstream-signaling pathways are important players in the control of adipocyte metabolism. It is surprising that expression of PKA regulatory and catalytic subunits for RI, RII, and C is highly detected from undifferentiated EMSC and has no change during differentiation. However, PKA regulatory subunit (RIIß) expression represents markers for fully differentiated adipocytes for both EMSC and S-V fractions. Further study will be necessary to determine C-associated fat cell differentiation in EMSC.
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3. EMSC cloning
Our previous data provided clear evidence of histochemical, morphological, and biochemical differentiation of EMSC into the osteogenic, chondrogenic, and adipogenic lineages. However, the differentiation into multiple lineages could be explained by the presence of mixed culture of precursor cells. To address this issue, we analyzed the differentiation potential of cloned EMSC isolated by the dilution plating technique. Figure 2
displays a typical morphological pattern of undifferentiated EMSC clones (Fig. 2A
) and clones differentiated into osteogenic (Fig. 2B
), adipogenic (Fig. 2C
), and chondrogenic (Fig. 2D
) lineages. The differentiation toward particular lineage was confirmed by the expression of lineage-specific genes.
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Osteogenesis was characterized by strong expression of osteocalcin gene in 4 clones (Fig. 2E
). The size of mRNA for osteocalcin was consistent with that found with RNA extracted from the native tissues (limb). Three of 4 clones differentiated into the chondrogenic lineages evident by procollagen II expression (Fig. 2G
).
The adipogenic potential of EMSC clones was revealed morphologically by lipid drops accumulation (Fig. 2C
) and at the molecular level by expression of PPAR, aP2, and LPL (Fig. 2F
). Although all 4 clones expressed adipogenic marker genes, PPAR, aP2, and LPL expression varied among clones. The strongest adipogenic expression was observed in clone 3, where expression of marker genes PPAR, aP2, and LPL was prominent (Fig. 2C F
). None of the clones showed expression of differentiation markers in cells cultured under control conditions as observed in the uncloned mixed cell cultures (Fig. 2A
). Since cyclophillin was expressed in all of these cultures, there was no question about the quality of the RNA in the assays.
CONCLUSIONS AND SIGNIFICANCE
We have shown that cells isolated from the ears of different mouse strains have clonogenic potential and can differentiate into three mesenchymal lineages: adipocytes, osteocytes, and chondrocytes. Our previous study showed that the presence of EMSC is independent of the age of the animal and that they express the transcriptional factor Oct-4, a marker characteristic of undifferentiated embryonic stem cells (ES). EMSC undergo a facile adipogenic differentiation in vitro that is sustained beyond passage 5. The optimized culture conditions for adipogenic differentiation indicate that EMSC are an easily obtainable source of stem cells for the study of adipogenesis in a primary cell culture model.
The developmental process by which fibroblast-like preadipocytes acquire the structure and function of mature adipocytes has been extensively studied. Two in vitro cell culture models have been developed for the analysis of cellular and molecular mechanisms taking place during adipogenesis: 1) immortalized preadipocyte cell lines (3T3-L1 and 3T3-F442A) and 2) primary culture of the S-V fraction of fat depots. These studies have revealed that a cascade of transcription factors necessary for the differentiation of the preadipocyte to adipocyte are induced under appropriate hormonal stimulation and cell density. However, the early molecular events that promote commitment of mesenchymal precursor cells to the adipocyte lineage are not well established. Studies based on the immortalized embryo fibroblast lines C3H10T1/2 and 243 as well as adult stem cells, which are able to differentiate into mesodermal lineages, revealed that the commitment of adult stem cells into adipogenic lineage requires the same set of stimulation factors as the differentiation of preadipocytes to mature adipocytes. Similarly, our studies indicate that three basic components (insulin, dexamethasone, and isobutyl-methylxanthine) are sufficient to stimulate EMSC commitment/differentiation into the adipogenic lineage. However, we noted differences in the time needed to achieve 60–80% differentiation between our model and that of others. Human mesenchymal stem cells from human bone marrow require 18–25 days and multipotent human adipose tissue cells require14 days to differentiate into adipocytes. We found that EMSC developed morphological features characteristic of mature adipocytes after only 7–9 days in culture. Whether these differences reflect biological variation or culture conditions among different laboratories remains to be determined.
EMSC exemplify an excellent model for the study of adipogenesis if we agree that all connective tissues contain stem cells able to differentiate into lineages of mesodermal origin and are likely to be the source/origin of adipose tissues in severe obese individuals. Our recent observations (unpublished data) indicate that 4 mm diameter ear punches obtained from mice during standard procedure used for marking animals are sufficient for EMSC culture. This model allows one to conduct simultaneous in vivo and in vitro studies without the need to sacrifice animals or study adipogenesis in humans.
We conclude that ear cells have characteristics consistent with those expected of adult mesenchymal stem cells and that any normal or mutant strain of mice can readily be analyzed in culture for aspects of adipogenesis, chondrogenesis, and osteogenesis.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3204fje; doi: 10.1096/fj.04-3204fje
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