Induction of Adipocyte-Like Phenotype in Human Mesenchymal Stem Cells by Hypoxia - 《stemcells》2004-2005文献干细胞之家



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发表于 2009-3-5 10:35 |只看该作者 |倒序浏览 |打印

a Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Aarhus, Denmark;2 f4 n7 Y* H; a
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b Department of Endocrinology (KMEB), Odense University Hospital, Odense, Denmark+ |' D! G1 {; k& U9 W# T) V, S1 y. k- [
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Key Words. Adipogenesis ? Adult bone marrow stem cells ? Differentiation ? Hypoxia
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Correspondence: Vladimir Zachar, M.D., Ph.D., Laboratory for Stem Cell Research, Aalborg University, Gustav Wieds Vej 10, 8000 Aarhus C, Denmark. Telephone: 45-86127366; Fax: 45-86195415; e-mail: vladimir@lsr.aau.dk
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ABSTRACT

In everyday practice, the in vitro experiments are routinely being set up at an oxygen concentration in the gas phase that corresponds to that of the ambient air, and it is being overlooked that such conditions provide for oxygen levels that several-fold surpass those found under physiological conditions. Hypoxia has previously been shown to have a critical role in the development of placenta ; moreover, early human embryogenesis takes place in an environment with a very low oxygen tension . Adult stem cells residing in the bone marrow are similarly exposed to low oxygen tension. The mean oxygen concentration of the bone marrow has been reported to be approximately 7% . While, at present, it is not possible to measure spatial concentration of oxygen, mathematical models have indicated that there is a gradient across the marrow from the rather well-oxygenated sinuses to the relatively hypoxic areas near the bone endosteum . It has been proposed that the hematopoietic stem cells (HSCs) are maintained in an undifferentiated state in hypoxic niches in the bone marrow and that the expansion and differentiation of the stem cells occurs along an oxygen gradient . Recently, Danet et al.  demonstrated that culturing human HSCs under reduced oxygen conditions (1.5%) improved their survival and increased the number of bone marrow–repopulating cells. Furthermore, hypoxia has been shown to stimulate the formation of granulocytic-monocytic progenitors  and profoundly alter several characteristics of macrophages .* Y7 g  }4 `* U" l7 {7 a& A
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Bone marrow is also a place of residence of mesenchymal stem cells (MSCs), which are pluripotent cells with the potential for differentiating into several cell types, including adipocytes, chondrocytes, myocytes, and osteoblasts . It is noteworthy that oxygen has been shown to modulate the differentiation in at least some of these cell types, specifically osteoblasts and adipocytes. In particular, culturing rat MSCs at reduced oxygen tension (5%) increased their bone-forming potential , and in a study of adipogenesis by Yun et al. , hypoxia inhibited the conversion of mouse pre-adipocytes to adipocytes upon induction with a classical adipogenic cocktail. The initial data thus seem to indicate that oxygen has a regulatory role in at least some differentiation pathways.
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To obtain a better understanding of the effect of hypoxia on adipogenic conversion of MSCs, we subjected a human telomerase reverse transcriptase–immortalized human MSC (hMSC) line, hMSC-TERT , to oxygen concentrations ranging from 1%–21%, and we followed the accumulation of intracellular lipid and expression of marker genes associated with early and late stages of adipogenesis.& [+ `( \( x# D' a" O

MATERIALS AND METHODS; S6 |* W" H& Z& e4 ?
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Lipid Accumulation5 }: z8 q9 J& q$ l+ I: T
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To evaluate the effect of hypoxia on adipogenesis, hMSC-TERT cells were incubated at oxygen tensions covering the range from 1%– 6% for up to 72 hours. Only when they were cultured at oxygen tension corresponding to 1% did the cells respond with a distinct accumulation of lipid droplets. The changes of the phenotype became visible microscopically, as well as macroscopically, as soon as 24 hours after the initiation of treatment, as indicated by Oil red O staining (Fig. 1). Incubation of the cells at 2% oxygen and above (4% and 6% data not shown) did not appear to have any significant effect on lipid accumulation even after 72 hours of hypoxic exposure (Fig. 1). To obtain more information about the distribution of lipid load at the population level and the kinetics of lipid accumulation, the cells were stained with Nile red and subjected to flow cytometric analysis. As evidenced in Figure 2A, the lipid accumulation occurred in a symmetric fashion, corresponding to a normal distribution across the whole population of cells, at all time intervals. Furthermore, the quantitation of lipid accumulation in time by flow cytometry confirmed that when growing in 1% oxygen atmosphere, the onset of triglyceride accumulation was evident already after 24 hours and became statistically significant after 48 hours (Fig. 2B).( V) x+ i. i0 e: P, q

Figure 1. Effect of oxygen tension on lipid droplet formation in hMSC-TERT cells. The hMSC-TERT cells were cultured at ambient (21%) and hypoxic (2% and 1%) oxygen concentrations for up to 3 days. Lipid droplet accumulation was examined by a Hoffman modulation contrast microscopy after staining with Oil red O.4 p) @% Z8 O' U# g1 X
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Figure 2. Flow cytometric analysis of lipid accumulation and distribution in hMSC-TERT cells incubated at varying oxygen concentrations. The hMSC-TERT cells were incubated at 1%, 2%, and 21% oxygen concentrations for up to 3 days. At time 0, 8, 24, 48, and 72 hours of treatment, cells were fixed, stained with Nile red, and analyzed in a flow cytometer. (A): Results from a representative experiment of cells cultured for up to 72 hours at 1% oxygen concentrations. The shaded area represents cells prior to hypoxic treatment; open areas represent treated cells. (B): Lipid accumulation represented as the geometric mean obtained from flow cytometric analysis as a function of time and oxygen concentration. Each point represents the average of at least two experiments, each in duplicate, in which error bars denote standard error of mean. Asterisks indicate p * S$ ~5 K- E6 p1 A+ j& S$ l
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To obtain a better understanding of the differences between hypoxia-induced and rosiglitazone-induced lipid accumulation, the cells were incubated either at 1% oxygen partial pressure for 48 hours or with rosiglitazone-containing induction medium for 14 days, after which the triglyceride accumulation was visualized with Oil red O and fluorescent marker BODIPY (Fig. 3). The Oil red O staining revealed that the cells grown in hypoxia accumulated lipids in a remarkably different fashion from those incubated with rosiglitazone. In hypoxia, the lipids were found to form a diffuse pattern, whereas, in rosiglitazone-treated cells, the lipids were typically confined to discrete areas. The close-up micrographs of the BODIPY-stained cells further confirmed this observation. In hypoxia, all cells accumulated a small number of little lipid droplets, but in the foci of lipid accumulation, the rosiglitazone-treated cells contained an abundance of lipid inclusions that varied greatly in size, yet were for the most part significantly larger than those in hypoxia." \& X- \  @8 ]

Figure 3. Differential distribution of neutral lipids in hMSC-TERT cultures exposed to low oxygen tension or undergoing adipogenic differentiation. (A):The hMSC-TERT cells were cultured under ambient and hypoxic conditions, corresponding to 21% and 1% oxygen concentrations, respectively. After 72 hours, the cultures were stained with Oil red O and examined using a Hoffman modulation contrast microscopy or stained with BODIPY and Hoechst 33342 and subjected to an epifluorescence examination. (B): The hMSC-TERT cells were cultured under standard conditions or induced into adipogenesis by dexamethasone, isobutyl methylxanthine, insulin, indomethacin, and rosiglitazone. After 14 days, the cells were stained with Oil red O and fluorescent dyes.
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Pericellular Oxygen Tension9 t  p3 N* p$ n

The finding that the lipid accumulation in the hMSC-TERT cells was apparently controlled over a very narrow oxygen gradient warranted further investigation. Hence, to determine accurately at which point the lipid accumulation occurred, the pericellular oxygen tension was measured. The oxygen tensions in close vicinity of the cells were found almost 10-fold lower than above the medium (Table 1). Thus the lipid accumulation was induced at levels of pericellular oxygen concentration corresponding to 0.14% but, strikingly, the lipogenic process was efficiently abrogated by an increase in oxygen concentration as little as 0.1 percentage points. It is also noteworthy that when the cultures were transferred from ambient oxygen tension to hypoxia, the equilibrium between oxygen in the gas and liquid phases was reached after as soon as 1 hour (data not shown)." S, h2 \8 `: C: g; c* z# X: E. N
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Table 1. Correlation between atmospheric oxygen concentration and pericellular oxygen tension
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Adipocyte-Specific Gene Expression+ v! D! e. G5 c
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To determine the transcriptional phenotype of the cells grown in hypoxia and compare it with that of control cultures and genuine adipose tissue, a comprehensive group of adipocyte-specific genes was analyzed by a sensitive real-time RT-PCR (Table 2). First, the expression of the adipogenesis-related transcription factors ADD1/SREPB1c and PPAR-2 was evaluated. Surprisingly, no expression of these genes was detected in spite of the distinct lipid accumulation. Thereafter, we examined whether the lipid accumulation could be attributed to changes in the expression of some of the genes involved in lipid transport, storage, or metabolism. However, adipophilin, aP2, perilipin, and lipoprotein lipase were not expressed in the cells. Finally, when assessing the expression of adipocytokines, including adiponectin, leptin, and PGAR, we found that PGAR was upregulated 12-fold in 1% O2 (12-fold). The expression of the other two adipocytokines, adiponectin and leptin, however, could not be detected.

Table 2. Expression of adipocyte-specific genes in hMSCs after exposure to 72 hours of hypoxia or 14 days of rosiglitazone treatment

To confirm that the cell line could express the adipocyte-specific markers under appropriate culture conditions, the rosiglitazone-treated cells were subjected to the same analysis. As indicated in Table 2, the results from real-time RT-PCR analysis corroborated the expression of all adipocyte-specific genes, except for adiponectin and leptin.0 z5 X, S/ U, a- {, W
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Although the hypoxia-induced transcription of PGAR decreased with increasing oxygen concentrations, the expression was still statistically significantly higher than the expression at 21% for all hypoxic conditions (Fig. 4). The transcription of GAPDH, which is responsive to hypoxia in several cell types, was also statistically significantly increased, but only in an atmosphere with less than 4% O2 (Fig. 4). This finding indicates that HIF-1 is stabilized in the hMSC-TERT cells below a specific level of oxygen concentration.3 L( X  f& U0 o- e
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Figure 4. Transcriptional regulation of GAPDH and PPAR-–induced angiopoietin-related gene (PGAR) by low oxygen. The hMSC-TERT cells were incubated at 1%, 2%, 3%, 4%, and 6% oxygen for 72 hours, after which RNA was harvested and analyzed by real-time reverse transcription polymerase chain reaction. The graph shows the fold regulation of GAPDH (filled bars) and PGAR (open bars) in hypoxic cells compared with cells cultured at ambient oxygen tensions (21%). The bars indicate the average of at least two independent experiments, each in duplicate (n = 4). Error bars designate standard error of the mean. Asterisks indicate p

Hypoxia and Rosiglitazone-Induced Expression of GAPDH and PPAR-2. V7 S3 H6 e& L' A
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The effect of hypoxia and rosiglitazone treatment on protein levels of GAPDH and PPAR-2 in hMSC-TERT cells was determined by Western blotting (Fig. 5). The analysis revealed that treatment with 1% oxygen for 72 hours resulted in accumulation of GAPDH, whereas treatment with rosiglitazone did not increase GAPDH protein levels above the basal expression. Furthermore, only cells treated with rosiglitazone accumulated PPAR-2, thus confirming the findings by real-time RT-PCR.

Figure 5. Induction of GAPDH and PPAR-2 proteins by low oxygen and rosiglitazone. The hMSC-TERT cells were cultured in normal growth medium at 21% or 1% oxygen for 72 hours, after which the whole cell lysates were analyzed by immunoblotting. For adipogenic induction, the normal growth medium was supplemented with dexamethasone, isobutyl methylxanthine, insulin, indomethacin, and rosiglitazone, and the analysis was carried out after 14 days. Abbreviations: GAPDH, glycer-aldehyde-3-phosphate dehydrogenase; PPAR-2, peroxisome proliferator-activated receptor 2.
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DISCUSSION- X7 R8 q' m4 [- E
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hMSCs are capable of differentiating into various cell types, including adipocytes, myocytes, chondrocytes, and osteocytes. We have observed that hMSCs incubated under hypoxic conditions (1% oxygen) form adipocyte-like phenotype with cytoplasmic accumulation of lipid. In spite of increased levels of the PPAR-–induced angiopoietin-related gene (PGAR) transcripts, the accumulation of lipids was not accompanied by increased transcription of adipocyte-specific genes such as ADD1/SREBP1c, PPAR-2, lipoprotein lipase, aP2, leptin, perilipin, and adipophilin. In conclusion, it appears that under specific hypoxic conditions hMSCs may acquire adipocyte-mimicking morphology in the absence of true adipogenic conversion.
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