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Keywords:

  • Stem cell antigen 1;
  • Mesenchymal stem cells;
  • Adipogenesis;
  • Differentiation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Summary
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

Hyperplasia is a major contributor to the increase in adipose tissue mass that is characteristic of obesity. However, the identity and characteristics of cells that can be committed into adipocyte lineage remain unclear. Stem cell antigen 1 (Sca-1) has been used recently as a candidate marker in the search for tissue-resident stem cells. In our quest for biomarkers of cells that can become adipocytes, we analyzed ear mesenchymal stem cells (EMSC), which can differentiate into adipocytes, osteocytes, chondrocytes, and myocytes. Our previous studies have demonstrated that EMSC abundantly expressed Sca-1. In the present study, we have analyzed the expression of adipogenic transcription factors and adipocyte-specific genes in Sca-1-enriched and Sca-1-depleted EMSC fractions. Sca-1-enriched EMSC accumulated more lipid droplets during adipogenic differentiation than Sca-1-depleted. Similarly, EMSC isolated from Sca-1−/− mice displayed reduced lipid accumulation relative to EMSC from wild-type controls (p < .01). Comparative analysis of the adipogenic differentiation process between Sca-1-enriched and Sca-1-depleted populations of EMSC revealed substantial differences in the gene expression. Preadipocyte factor 1, CCAAT enhancer-binding protein (C/EBP) β, C/EBPα, peroxisome proliferator-activated receptor γ2, lipoprotein lipase, and adipocyte fatty acid binding protein were expressed at significantly higher levels in the Sca-1-enriched EMSC fraction. However, the most striking observation was that leptin was detected only in the conditioned medium of Sca-1-enriched EMSC. In addition, we performed loss-of-function (Sca-1 morpholino oligonucleotide) experiments. The data presented here suggest that Sca-1 is a biomarker for EMSC with the potential to become functionally active adipocytes.

Disclosure of potential conflicts of interest is found at the end of this article.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Summary
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

Author contributions: J.S.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; J.M.G.: conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript; J.A.M.: collection and assembly of data, final approval of manuscript; B.G.-K.: conception and design, administrative support, collection and assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript.

We have previously established and characterized a primary culture of mouse ear mesenchymal stem cells (EMSC) that are isolated from the external murine ears [1]. These cells have the characteristics of stem cells, including the ability to self-renewal and to differentiate into various types of cells at the clonal level [1, [2]3]. Immunophenotype analyses of EMSC showed that undifferentiated EMSC are strongly positive for stem cell antigen 1 (Sca-1) and are negative for hematopoietic markers (CD45, CD4) [2]. Furthermore, we found robust accumulation of lipid droplets in Sca-1-enriched (Sca-1+), but not Sca-1-depleted (Sca-1) EMSC fractions exposed to an adipogenic induction medium. These observations have led us to postulate that Sca-1 plays a role in adipogenic differentiation.

A substantial body of literature links Sca-1 to stem cell function. Also known as a Ly-6A/E, Sca-1 is an 18-kDa glycosyl phosphatidylinositol-anchored cell surface protein initially identified as an antigenic marker of murine hematopoietic cells [4]. Sca-1 has been detected on bone marrow-derived mesenchymal stem cells [5, [6], [7]8], skeletal muscle stem cells [9], mammary epithelial stem cells [10], cardiac tissue [11], skin [12], muscle [13, [14], [15], [16], [17]18], kidney [19], testis [20], liver [21, 22], prostate [23, 24], and pulmonary endothelium [25]. Sca-1 is linked to the self-renewal of mesenchymal [26] and hematopoietic [27] progenitors in the bone marrow and myogenic stem cells [28]. In addition, it has been reported that skeletal muscle-derived and bone marrow-derived mesenchymal stem cells expressing Sca-1 can differentiate into cardiomyocytes in vivo [6] and contribute to muscle regeneration [29], respectively. Myogenic and endothelial cell progenitors identified in the interstitial spaces of murine skeletal muscle, which are strongly positive for Sca-1, display the potential to differentiate into adipocytes, endothelial cells, and myogenic cells [18]. Moreover, a population of Sca-1+ cells has been identified in neonatal mouse skin that expresses adipocyte markers [30]. These observations are consistent with our EMSC observations.

To test our hypothesis that Sca-1 plays a role in adipogenic differentiation, we have compared the adipogenic capacity of Sca-1-enriched versus Sca-1-depleted populations of EMSC using both antibody-based sorting and loss-of-function experiments. As parameters for this in vitro evaluation, we have examined the expression of adipogenic transcription factors and adipocyte expressed genes, oil red O staining, BODIPY (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) staining, and leptin protein secretion.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Summary
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

Animals

C57BL/6J mice at ages of 3–6 weeks were used in the study. Experiments involving animals were approved by the Pennington Biomedical Research Center Institutional Animal Care and Use Committee in accordance with NIH guidelines. All procedures were designed to minimize the suffering of experimental animals. Mice were housed in a temperature- and humidity-controlled room (22°C ± 2°C and 30%–70%, respectively) with a 12-hour-light/12-hour-dark cycle (lights on at 6:00 a.m.) and were given ad libitum access to chow diet and tap water throughout the study. Mice were sacrificed by CO2 asphyxiation followed by cervical dislocation.

Cell Harvest and Culture

For isolation of EMSC, outer ears were excised, minced, and digested with collagenase type I (2 mg/1 ml; Worthington Biochemical, Lakewood, NJ, http://www.worthington-biochem.com) in a shaking bath for 1 hour at 37°C. The cell suspension was filtered through a 70-μm cell strainer (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) followed by centrifugation (360g, 5 minutes, room temperature). Pelleted cells were resuspended in 1 ml of red blood lysis buffer (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) and centrifuged as described above. The isolated cells were plated in 100-mm Petri dishes (P0) in Dulbecco's modified Eagle's medium/Ham's F-12 medium (DMEM/F12; Invitrogen) supplemented with 1% antibiotic solution and 15% fetal bovine serum (FBS; Invitrogen). Subconfluent primary cultures were trypsinized (0.05% trypsin/0.53 mM EDTA; Life Technologies, New York, http://www.lifetech.com), followed by immunomagnetic cell sorting.

Sca-1 Magnetic Sorting

Magnetic labeling cell sorting with anti-Sca-1 immunomagnetic MicroBeads (Miltenyi Biotec, Auburn, CA, http://www.miltenyibiotec.com) was used according to the manufacturer's protocol to sort Sca-1-enriched and Sca-1-depleted fractions of isolated ear mesenchymal stem cells. Briefly, up to 107 cells (P0) were initially labeled with 10 μl of anti-Sca-1-fluorescein isothiocyanate (FITC) followed by magnetic labeling with 20 μl of anti-FITC MicroBeads. The cell suspension was then transferred to a magnetic cell sorting (MACS) column placed in the magnetic field of a MACS separator. Unlabeled (Sca-1) cells were eluted with a buffer (phosphate-buffered saline [PBS] with 0.5% bovine serum albumin and 2 mM EDTA). The column was removed from the separator, and retained Sca-1+ cells were flushed out with the buffer. The purity of each fraction was analyzed using a flow cytometer (Becton, Dickinson and Company, San Jose, CA, http://www.bd.com) as previously described [2].

Cell Doubling Assay

Cells were seeded in a 96-well plate at a density of 5 × 104 cells per well. On days 1 and 4 the cells were fixed with 10% formalin for 1 hour at room temperature followed by staining with 300 nM 4,6-diamidino-2-phenylindole (DAPI) (Invitrogen) for 10 minutes at room temperature. Stained nuclei were visualized using a Nikon Eclipse TE2000-U (Nikon Instruments, New York, http://www.nikon.com) inverted microscope equipped with a CoolSnap camera (Nikon Instruments). Images of random fields were acquired with Metamorph imaging software (Molecular Devices Corp., Sunnyvale, CA, http://www.moleculardevices.com), and cells were counted using image analysis software (ImageJ; http://rsb.info.nih.gov/ij). Cell doubling times were calculated according to the following formula [31]:

  • equation image

where DT is the cell doubling time, CT is the cell culture time, Nf is the final number of cells, and Ni is the initial number of cells.

In Vitro Adipogenic Differentiation

Cells were replated in 6- or 12-well culture plates (Corning Enterprises, Corning, NY, http://www.corning.com) at a density of 104 cells per cm2 and maintained in complete medium until confluent (considered day 0). Thereafter, the cells were exposed to an adipogenic induction medium containing DMEM/F12, 5% FBS, 1% antibiotic solution, 0.5 mM isobutylmethylxanthine, 1.7 μM insulin, and 1 μM dexamethasone for 2 days (adipogenic medium I). For the next 7 days, medium was changed to DMEM/F12 supplemented with 5% FBS, 1% antibiotic solution, 17 nM insulin, and 2 μM thiazolidinedione (adipogenic medium II). On days 0, 3, 6, and 9, cells were harvested for RNA and protein purification, whereas culture medium were collected for leptin assay.

Loss-of-Function Experiment

Sca-1 morpholino oligonucleotides (Sca-1 MO) used in this study were designed and synthesized by Gene Tools, LLC (Philomath, OR, http://www.gene-tools.com), on the basis of the cDNA sequence of Sca-1 (Ly6a/E; GI:31981636). The morpholino sequence against the Sca-1 mRNA was 5′CTT TGT AGT GTG AGA AGT GTC CAT C3′. An irrelevant morpholino (standard control MO) and FITC-labeled standard control MOs were also purchased from Gene Tools. The MOs were prepared at a stock concentration of 500 μM. EMSC were cultured in six-well plates in DMEM/F12 with 15% FBS. Subconfluent cells were treated with morpholino oligos and Endo-Porter in DMEM/F12 with 5% FBS (day −1; Fig. 2A). At day 0, adipogenic medium I was added to the culture. After 48 hours, medium was changed to adipogenic medium II. At day 3, morpholino oligos and Endo-Porter were added a second time to the culture. Every 3rd day during this process, selected cultures were removed for biochemical/morphological analysis by staining with oil red O. In addition, total RNA and protein were isolated from similar cultures to measure marker gene expression by real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) and Western blot, respectively.

RNA Isolation and Quantitative Real-Time Reverse Transcriptase Polymerase Chain Reaction

Total RNA was extracted using Trizol (Invitrogen) and column-purified with RNeasy and RNase-Free DNase kits (Qiagen, Valencia, CA, http://www1.qiagen.com). cDNA synthesis was performed with 500 ng of total RNA using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). Endogenous mRNA levels for Sca-1, preadipocyte factor 1 (Pref-1), Wnt-10b, CCAAT enhancer-binding protein (C/EBP) β, CEBP/δ, C/EBPα, peroxisome proliferator-activated receptor (PPAR) γ2, adipocyte fatty acid binding protein (aP2), lipoprotein kinase (Lpl), and leptin were measured with TaqMan Gene Expression Assays (Applied Biosystems). Reactions were performed in MicroAmp Optic 384-well reaction plates (Applied Biosystems) using the ABI Prism 7900 sequence detection system (Applied Biosystems) under conditions of 2 minutes at 48°C, 10 minutes at 95°C, and then 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. The quantitative real-time polymerase chain reaction (qRT-PCR) was performed in duplicate for each sample, and each run included a standard curve, nontemplate control, and negative RT control. Levels of gene expression were quantified relative to the level of hypoxanthine phosphoribosyltransferase 1 (HPRT1) using a standard curve method. HPRT1 was chosen as the internal control gene on the basis of published [32] and our own data. Our preliminary qRT-PCR experiments did not show a significant difference in the mean levels of HPRT1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression between day 0 and day 9 of adipogenic differentiation (data not shown). However, since the SD of the HPRT1 gene was much tighter than that of GAPDH, we selected HPRT1 as our housekeeping gene for following studies.

Western Blot

Total cell lysates were prepared by adding 400 μl of RIPA buffer containing protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktails I and II (Sigma-Aldrich). Total protein (40 μg) was separated on 20% SDS-polyacrylamide gels under nonreducing conditions and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, http://www.millipore.com). The blots were then incubated with monoclonal antibodies against Sca-1 (eBioscience Inc., San Diego, http://www.ebioscience.com) diluted to a concentration of 1:100. Bands were visualized using the Odyssey imaging system (LI-COR Biosciences, San Diego, http://www.licor.com) with fluorescent (IRDye800TM or Cy5.5)-labeled secondary antibodies according to the manufacturers' protocols.

Leptin Concentration in Medium

Adipogenic differentiation medium were collected on days 0, 3, 6, and 9. Leptin concentrations were determined using the DuoSet enzyme-linked immunosorbent assay development kit (R&D Systems Inc., Minneapolis, http://www.rndsystems.com) according to manufacturer's protocol. Briefly, a 96-well plate was coated with 100 μl of capture antibody and incubated overnight at room temperature. Next, 100 μl of detection antibody was added to 100 μl of sample or standard. After a 2-hour incubation, 100 μl of working dilution of streptavidin-horseradish peroxidase was added for 20 minutes followed by incubation with 100 μl of substrate solution. The reaction was terminated with 50 μl of stop solution. The optical density of each well was read at 450 nm, with wavelength correction at 540 nm. The concentration was calculated with a standard curve.

Oil Red O Staining

Differentiated cells were fixed for 1 hour in 10% formalin at room temperature and later stained for lipid accumulation for 20 minutes with oil red O. Cells were washed three times with water and observed under a phase-contrast microscope. The dye retained by cells was eluted with isopropanol followed by absorbance measurements at 500 nm [33].

BODIPY/DAPI Staining

Differentiated cells were fixed with 10% formalin for 1 hour at room temperature followed by staining with 300 nM DAPI/10 μg/ml BODIPY 493/503 in PBS for 20 minutes at room temperature. After washing three times for 5 minutes each in PBS, cells were imaged with a Plan Fluor DL ×10 objective using a Nikon Eclipse TE2000-U inverted microscope equipped with a CoolSnap camera. Images of random areas were captured and stored with Metamorph imaging software. Total area of BODIPY-stained lipid droplets (relative to a number of DAPI-stained nuclei) was measured using ImageJ image analysis software. Results are expressed as pixels per cell.

Sca-1 KO Versus C57BL/6J Experiment

Sca-1 knockout (KO) breeding pairs (C57BL/6J background [27]) were a kind gift from William L. Stanford (Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada). Three-week-old Sca-1 KO (n = 10) and C57BL/6J (n = 10) mice were used for experiment. EMSC were isolated from 4-mm ear punches obtained during the standard procedure used for marking live animals. Cells were harvested and cultured as described previously [2]. In vitro adipogenic differentiation was performed on cells from passage 2 seeded in a 96-well plate. The cells were exposed to an adipogenic induction medium followed by BODIPY/DAPI staining and image analysis as described above.

Statistical Analysis

Data expressed as mean ± SD were analyzed with a two-tailed Student's test. A value of p < .05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Summary
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

Our previous study revealed that EMSC undergo robust adipogenic differentiation [1]. In addition, flow cytometric analyses showed that undifferentiated EMSC are highly positive (82.77% ± 7.8%) for Sca-1 [2]. The present experiments were undertaken to test the hypothesis that Sca-1 plays a role in adipogenic differentiation.

Sca-1+ Cells Display Enhanced Adipocyte Differentiation Capacity

Flow cytometric analysis of immunomagnetically sorted cells revealed that 96.36% ± 3.52% of EMSC in the Sca-1-enriched fraction and 12.06% ± 9.06% in the Sca-1-depleted fraction expressed Sca-1 (n = 18). Those data were confirmed by parallel analyses of Sca-1 mRNA content in both EMSC fractions (Fig. 1). Our next experiment asked whether there are differences in the proliferation rate of Sca-1-enriched versus Sca-1-depleted EMSC fractions. The doubling times calculated for both fractions showed no statistical difference (2.72 ± 1.08 vs. 3.19 ± 1.33, respectively; p = .5189; Fig. 2). Additional studies compared the adipogenic capacity of Sca-1-enriched and Sca-1-depleted fractions cultured under identical conditions. Following the induction of adipogenic differentiation, the expression of Sca-1 in both fractions was downregulated (Fig. 1). The mRNA levels of negative regulators of adipocyte differentiation, Pref-1 (Fig. 3A) and Wnt-10b (Fig. 3B), were elevated in the undifferentiated EMSC (day 0) and downregulated following adipogenic stimulation (days 3, 6, and 9). The expression of the transcription factors C/EBPβ (Fig. 3C) and C/EBPδ (Fig. 3D) showed a similar pattern, with the expression highest at day 0 and decreasing during the course of the differentiation process (days 3–9). In contrast, the mRNAs encoding the adipogenic C/EBPα and PPARγ2 transcription factors were at low abundance on day 0 but were significantly upregulated following adipogenic stimulation (Fig. 3E, 3F). The mRNA encoding late markers of adipogenic differentiation, Lpl and aP2, showed a similar pattern of expression—low on day 0 followed by significant induction during subsequent days of differentiation (Fig. 3G, 3H). Comparative analysis of the adipogenic differentiation process between Sca-1-enriched and Sca-1-depleted populations of EMSC revealed substantial differences in gene expression. Pref-1, C/EBPβ, C/EBPα, PPARγ2, Lpl, and aP2 were expressed at significantly higher levels in the Sca-1-enriched EMSC fraction. However, leptin expression displayed the most striking difference between the Sca-1-enriched and Sca-1-depleted EMSC fractions during adipogenic differentiation (Fig. 3I, 3J). Whereas the Sca-1+ fraction showed a time-dependent and substantial increase in leptin mRNA, the Sca-1-depleted fraction expressed leptin at low levels, with no change in its expression between day 3 and day 9 (Fig. 3I). Moreover, leptin secretion measured in conditioned media was detected only in Sca-1-enriched EMSC (Fig. 3J). To compare morphological changes and neutral lipid accumulation, cells from both fractions were stained with oil red O or BODIPY on days 0 and 9 of differentiation (Fig. 4A–4D). Based on oil red O staining, lipid accumulation was much denser in Sca-1-enriched cells (compare Fig. 4B and Fig. 4C), and this difference was statistically significant (0.72 ± 0.02 and 0.58 ± 0.09; p < .001; Fig. 4A). Based on BODIPY staining, the ratio of lipid droplets relative to the number of DAPI-stained nuclei was greater in the Sca-1-enriched EMSC population by a factor of 1.48 (p < .05; Fig. 4D).

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Figure Figure 1.. Real-time reverse transcription-polymerase chain reaction analysis for Sca-1 expression in Sca-1-enriched and Sca-1-depleted fractions of ear mesenchymal stem cells during the time course of adipogenic differentiation (***, p < .001; **, p < .01). Abbreviations: AU, arbitrary unit; Sca-1, stem cell antigen 1.

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Figure Figure 2.. Cell DT of cultured Sca-1-enriched and Sca-1-depleted fractions of ear mesenchymal stem cells. The values reflect the mean ± SD. Abbreviations: DT, doubling time; Sca, stem cell antigen.

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Figure Figure 3.. The Sca-1-enriched population of ear mesenchymal stem cell (EMSC) displays enhanced adipocyte differentiation capacity. (A–I): Gene expression profiles of Sca-1-enriched versus Sca-1-depleted fractions of EMSC during adipogenic differentiation. (J): Leptin protein secretion in cultured media. *, p < .05; **, p < .01; ***, p < .001. Abbreviations: AU, arbitrary unit; C/EBP, CCAAT enhancer-binding protein; PPAR, peroxisome proliferator-activated receptor; Pref-1, preadipocyte factor 1; Sca-1, stem cell antigen 1.

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Figure Figure 4.. Adipogenic potentials of Sca-1-enriched versus Sca-1-depleted ear mesenchymal stem cells (EMSC) (A–D) and C57BL/6J versus Sca-1−/− EMSC (E). (A): Spectrophotometric analysis of oil red O staining. (B, C): Phase-contrast micrographs of Sca-1-enriched (B) and Sca-1-depleted (C) cells at day 9 of adipogenic differentiation. Shown are image analyses of area of BODIPY-stained lipid droplets adjusted to the cell number of Sca-1-enriched versus Sca-1-depleted cells (D), and C57BL/6J versus Sca-1−/− EMSC (E). *, p < .05; **, p < .01; ***, p < .001. Abbreviations: OD, optical density; Sca-1, stem cell antigen 1.

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The lower adipogenic potential of the Sca-1-negative fraction has also been confirmed by using EMSC isolated from our recently established Sca-1−/− mouse colony (C57BL/6J genetic background). From our limited number of Sca1 KO animals we collected ear punches, and we and used these 4-mm-diameter tissue samples to isolate EMSC from live animals [2]. The EMSC isolated from Sca-1−/− mice and exposed to adipogenic cocktails showed a significantly lower (p < .01) accumulation of BODIPY-stained lipid droplets relative to DAPI-stained nuclei in comparison to EMSC derived from wild-type C57BL/6J mice (Fig. 4E).

Sca-1 Morpholino Knockdown Has No Effect on EMSC Adipogenic Differentiation

Morpholino oligonucleotides (MO) do not cause degradation of their RNA targets but reduce their biological activity through post-transcriptional mechanisms. Consequently, Western blot analyses of protein levels, rather than RT-PCR analyses of mRNA levels, are the most suitable assay of MO activity and effectiveness. As a first step to estimate the efficacy of our Sca-1 MO, we performed Western blot analysis of Sca-1 expression during EMSC adipogenic differentiation (Fig. 5A). The temporal analysis revealed a low expression of Sca-1 protein on day −1 in nonconfluent cells that increased on day 0 at confluence and was sustained during the subsequent first 2 days of adipogenic differentiation. A gradual decrease in Sca-1 protein expression was observed between day 3 and day 9 of differentiation.

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Figure Figure 5.. Sca-1 MO downregulate Sca-1 expression in ear mesenchymal stem cell (EMSC). (A, B): Western blot analysis of Sca-1 expression during EMSC adipogenic differentiation. (A): Untreated EMSC. (B): MO-treated EMSC. Shown are Sca-1 MO at days 2 and 5 and Ctr MO at day 2. Lane 1, 1 μM Sca-1 MO + 2 μM Endo-Porter; lane 2, 1 μM Sca-1 MO + 6 μM Endo-Porter; lane 3, 10 μM Sca-1 MO + 2 μM Endo-Porter; lane 4, 10 μM Sca-1 MO + 6 μM Endo-Porter; lane 5, 1 μM Ctr MO + 2 μM Endo-Porter; lane 6, 1 μM Ctr MO + 6 μM Endo-Porter; lane 7, 1 μM Sca-1 MO + 2 μM Endo-Porter; lane 8, 1 μM Sca-1 MO + 6 μM Endo-Porter. (C): Western blot analysis of Sca-1 expression on days 0, 3, 6, and 9 of adipogenic differentiation in Sca-1 MO-treated, Ctr MO-treated, and control/untreated cultures. (D): Gene expression profiles of morpholino-treated EMSC. (E): Spectrophotometric analysis of lipid droplet accumulation indicated by staining with oil red O. Abbreviations: AU, arbitrary unit; Ctr MO, control morpholino; PPAR, peroxisome proliferator-activated receptor; Pref-1, preadipocyte factor 1; Sca-1, stem cell antigen 1; Sca-1 MO, Sca-1 morpholino oligonucleotides.

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To estimate the optimal efficacy of Sca-1 MO, EMSC at subconfluency (day −1) or day 3 of differentiation were treated with increasing concentrations of MO and its delivery component (Endo-Porter; Gene Tools) (Fig. 5B). On the basis of Western blot analysis of Sca-1 expression and the microscopic appearance of cells, we identified the optimal concentrations of Sca-1 MO and Endo-Porter to be 1 and 6 μM, respectively (Fig. 5B). Once the effectiveness of the Sca-1 MO was established, we further tested the role of Sca-1 in adipogenic differentiation by determining whether blocking of Sca-1 expression in EMSC alters their adipogenic capacity. EMSC were treated with Sca-1 MO or a scrambled control MO on days −1 and 3 of the differentiation procedure. The Western blot data (Fig. 5C) documented inhibition of Sca-1 protein expression in Sca-1 MO-treated cells on days 3, 6, and 9 of differentiation. However, spectrophotometric analysis of oil red O staining (Fig. 5E) and adipogenic morphology (data not shown) showed no differences between Sca-1 MO-treated and control scrambled MO-treated cultures. The differentiation of EMSC into adipocytes was verified by qRT-PCR of Pref-1, Wnt-10b, PPARγ2, aP2, Lpl, and leptin in total RNA from control MO-treated and Sca-1 MO-treated cultures (Fig. 5D). Gene expression analyses revealed no differences between Sca-1 MO-treated and control MO-treated cultures.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Summary
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

The present study shows that Sca-1-enriched and Sca-1-depleted populations of EMSC differ in their capacity to differentiate into adipocytes. This conclusion is based on quantitative measures of neutral lipid accumulation, as well as on the expression of mRNAs encoding adipogenic transcription factors. Furthermore, a disparity in leptin secretion has been noted.

The epidermal growth factor-related surface protein Pref-1, also known as δ-like protein 1 (dlk-1), plays a role in maintaining preadipocytes in their undifferentiated state [34, 35]. Pref-1 expression is repressed when preadipocytes are induced to differentiate, and overexpression of the gene inhibits adipocyte differentiation [36]. Similarly, Wnt-10b has been demonstrated to inhibit adipogenic differentiation [37, 38], and disruption of its extracellular signaling results in spontaneous adipocyte conversion in vitro. In the present study, the expression of both Pref-1 and Wnt-10b mRNA was suppressed upon induction of EMSC differentiation.

Members of the C/EBP family of transcription factors have long been recognized as serving a critical role in adipogenic differentiation [39]. In our study, the C/EBP expression pattern of the EMSC mirrored those reported in studies of 3T3-L1 preadipocytes—expression of C/EBPβ and C/EBPδ preceded that of both C/EBPα and PPARγ [40, 41], which together induce expression of the complement of genes necessary to create the adipocyte phenotype.

PPARγ, a member of the peroxisome proliferator-activated receptor family of nuclear hormone receptors, is recognized as serving a central role in the regulation of adipogenesis [42, 43]. PPARγ2 is the predominant isoform found in adipocytes, and its expression, along with that of late adipogenic markers (Lpl and aP2), was upregulated during the course of differentiation.

The most significant difference between Sca-1-enriched and Sca-1-depleted EMSC was their expression of leptin, at both the mRNA and protein level. Despite the fact that Sca-1-depleted EMSC accumulated lipid droplets, their leptin mRNA content remained very low following adipogenic induction, and their secreted leptin protein level was undetectable. These observations may suggest that Sca-1 plays a positive role in leptin secretion; however, EMSC depleted of Sca-1 by morpholino treatment did not show downregulation of leptin secretion. Thus, the data suggest the existence of two different types of stem cells/preadipocyte precursors. The first exhibits Sca-1 positivity and gives rise to fully functional adipocytes, whereas the second is Sca-1-negative and less capable of adipocyte differentiation. Studies from the literature support this hypothesis since preadipocytes isolated from different fat depots display different adipogenic potential [44, 45]. For example, Tchkonia et al. [46] showed that there are substantial differences between preadipocytes from mesenteric and omental visceral depots, based on their gene expression profiles and capacity for replication and adipogenesis. Likewise, substantial differences exist in leptin and adiponectin gene expression between porcine intramuscular and subcutaneous adipocytes [47]. Our observations regarding the enhanced adipogenic capacity of Sca-1-expressing EMSC are consistent with previously published data. Colony-forming unit adipocyte assays have determined that Sca-1−/− bone marrow-derived cells formed 50% fewer colonies compared with Sca-1+/+ cultures [26]. An independent study showed diverse, age-dependent adipogenic differentiation of mesenchymal progenitors derived from Sca-1−/− versus Sca-1+/+ mice. Bone marrow-derived cells from the Sca-1+/+ mice exhibited significantly greater numbers of spontaneously differentiating oil red O-stained colonies at the ages of 7 and 9 months. In contrast, younger mice (3 and 5 months of age) showed no significant difference; marrow-derived cells from both Sca-1+/+ and Sca-1−/− displayed a limited capacity to form adipogenic colonies in the absence of adipogenic stimuli [48]. Although our study used young mice (3–6 weeks of age), we added adipogenic inductive cocktails to the EMSC at confluence. Thus, both exogenous and tissue-specific paracrine factors may account for the difference in our current findings and those in the literature. Consistent with our report, a higher adipogenic potential was reported in the Sca-1+ fraction of cells isolated from fetal mouse calvaria; this study likewise used an adipogenic inductive cocktail over a 21-day period [49]. In addition, significantly higher PPARγ expression was determined in Sca-1+ calvarial cells compared with Sca-1 cells [44].

Whereas Sca-1 cells may have a limited adipogenic capacity, they can differentiate along other lineage pathways. Steenhuis et al. showed that cells from fetal mouse calvaria within the Sca-1 fraction have higher chondrogenic and osteogenic potential relative to the Sca-1+ fraction [49]. These authors observed positive alkaline phosphatase staining primarily in the Sca-1 fraction following chondrogenic differentiation, and only Sca-1 cells exhibited mineralization when exposed to osteogenic medium [49]. Likewise, Sca-1 cells have been found to display more efficient myogenic differentiation [28]. C2C12 myogenic Sca-1 cells formed myotubes robustly, whereas myotube formation by Sca-1+ cells was not observed [28]. These findings and parallel gain-of-function/loss-of-function experiments have led to the suggestion that Sca-1 expression not only defines a subpopulation of muscle cells that are restricted to the myogenic lineage but also serves a functional role by regulating myoblast proliferation and myogenic differentiation.

Summary

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Summary
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

Sca1 is an intriguing marker that selects for EMSC with enhanced adipogenic potential. Additional studies, using the Sca1−/− murine model, will explore what role, if any, this surface antigen plays in regulating EMSC adipocyte commitment and differentiation.

Disclosure of Potential Conflicts of Interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Summary
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

The authors indicate no potential conflicts of interest.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Summary
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

We thank Marilyn Dietrich for flow cytometry analysis, Tamra Mendoza for assisting with animal husbandry, and William Stanford for providing the Sca-1−/− murine model. This work was supported by NIH Grant R01 1 P20 RR021945 COBRE.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Summary
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References