AbstractThe bone marrow microenvironment comprises multiple cell niches derived from bone marrow mesenchymal stem cells. However, the molecular mechanism of bone marrow mesenchymal stem cell differentiation is poorly understood. The transcription factor GATA2 is indispensable for hematopoietic stem cell function as well as other hematopoietic lineages, suggesting that it may maintain bone marrow mesenchymal stem cells in an immature state and also contribute to their differentiation. To explore this possibility, we established bone marrow mesenchymal stem cells from GATA2 conditional knockout mice. Differentiation of GATA2-deficient bone marrow mesenchymal stem cells into adipocytes induced accelerated oil-drop formation. Further, GATA2 loss- and gain-of-function analyses based on human bone marrow mesenchymal stem cells confirmed that decreased and increased GATA2 expression accelerated and suppressed bone marrow mesenchymal stem cell differentiation to adipocytes, respectively. Microarray analysis of GATA2 knockdowned human bone marrow mesenchymal stem cells revealed that 90 and 189 genes were upregulated or downregulated by a factor of 2, respectively. Moreover, gene ontology analysis revealed significant enrichment of genes involved in cell cycle regulation, and the number of G1/G0 cells increased after GATA2 knockdown. Concomitantly, cell proliferation was decreased by GATA2 knockdown. When GATA2 knockdowned bone marrow mesenchymal stem cells as well as adipocytes were cocultured with CD34-positive cells, hematopoietic stem cell frequency and colony formation decreased. We confirmed the existence of pathological signals that decrease and increase hematopoietic cell and adipocyte numbers, respectively, characteristic of aplastic anemia, and that suppress GATA2 expression in hematopoietic stem cells and bone marrow mesenchymal stem cells.
Bone marrow mesenchymal stem cells (BM-MSC) are selfrenewing precursor cells that differentiate into bone, fat, cartilage, and stromal cells of the bone marrow, thereby forming a microenvironment that maintains hematopoietic stem cells.1 Accumulating evidence indicates the importance of the bone marrow microenvironment during hematopoietic cell development. Increased adipogenesis in the bone marrow negatively affects hematopoietic activity,32 whereas the osteoblastic niche supports hematopoietic stem cell function by activating Notch signaling.4 Therefore, precise regulation of BM-MSC differentiation into various lineages maintains hematopoiesis.
Preadipocytes derived from MSC mature into adipocytes through a complex process involving numerous extracellular factors as well as transcription factors.51 Studies conducted on preadipocyte cell lines, such as mouse 3T3-L1 and 3T3-F442A, have uncovered the CCAAT/enhancer binding protein (C/EBP) family of transcription factors and the peroxisome proliferator-activated receptor γ (PPARγ) as key proadipogenic regulators.76 During preadipocyte–adipocyte differentiation, the expression of C/EBPβ and C/EBPδ initially increases, which subsequently activates the expression of C/EBPα and PPARγ, leading to the induction of genes involved in adipocyte function.98 However, the mechanism of differentiation of BM-MSC into adipogenic progenitors and ultimately into mature adipocytes in the bone marrow remains to be elucidated.
GATA2, a transcription factor critically required in the genesis and/or function of hematopoietic stem cells (HSC),1310 is expressed in various hematopoietic and non-hematopoietic tissues, including HSC, multipotent hematopoietic progenitors, erythroid precursors, megakaryocytes, eosinophils, mast cells, endothelial cells, and specific neurons.16141211 GATA2 is expressed by preadipocytes and BM-MSC and plays a central role in the control of adipogenesis.171613 GATA2 overexpression in a mouse preadipocytic stromal cell line induces resistance to adipocyte differentiation, whereas GATA2 knockdown accelerates adipocyte differentiation,17 implying that GATA2 functions to arrest preadipocyte differentiation. Although GATA2 may suppress transcription of C/EBP and PPARγ in preadipocytes,1816 the molecular mechanism by which GATA2 controls adipocyte differentiation remains unclear.
Aplastic anemia is characterized by decreased HSC and fatty marrow replacement. Moreover, GATA2 expression is decreased in CD34-positive cells in aplastic anemia,2019 Because BM-MSC express GATA2, it is possible that the signal that downregulates GATA2 expression in HSC may also suppress its expression in BM-MSC in aplastic anemia, thereby resulting in fewer HSC and an impaired microenvironment, which could support hematopoiesis. To test this hypothesis, we assessed the role of GATA2 during differentiation from BM-MSC.
Generation of bone marrow mesenchymal stem cells
To generate mouse BM-MSC, bone marrow cells from GATA2 conditional knockout mice were cultured in MesenCult MSC Basal Medium supplemented with 20% MSC stimulatory supplements (Stem Cell Technologies). The BM-MSC were transfected with the retroviruses expressing iCre to delete the DNA binding domain of GATA2 by inducing the Cre-loxP system.2221
To generate human BM-MSC, bone marrow mononuclear cells from healthy donors were cultured with Dulbecco’s modified Eagle’s medium (Life Technologies) supplemented with 20% fetal bovine serum (Life Technologies), 10 ng/mL basic fibroblast growth factor (PeproTech), 10 mM HEPES (Life Technologies), and 100 μg/mL penicillin/streptomycin (Invitrogen).2523 Established BM-MSC were used until the seventh generation.
The study was approved by the ethical committee of Tohoku University Graduate School of Medicine. Clinical samples were collected after obtaining written informed consent. The ethics policies of the Declaration of Helsinki were followed.
Characterization of bone marrow mesenchymal stem cells
BM-MSC immunophenotypes were determined using a FACSAria II (BD). To induce differentiation into adipocytes, human Mesenchymal Stem Cell Adipogenic Differentiation Medium (Lonza) was used. After 12–16 days, morphological changes were assessed using an inverted microscope. Typical adipocytes were stained with Oil Red O.2 The area of mature adipocytes was determined using HistoQuest software (Novel Science).
Quantitative reverse transcriptase polymerase chain reaction analysis and transcription profiling
Quantitative reverse transcriptase polymerase chain reaction analysis (RT-PCR) was performed as previously described.26 Primer sequences are available upon request.
For transcription profiling, the Human Genome U133 Plus 2.0 Array was used (Affymetrix). Gene ontology analysis was conducted using the DAVID bioinformatics program (http://david.abcc.ncifcrf.gov/).
Short interfering RNA-mediated knockdown
Anti-GATA2 and control short interfering RNA (siRNA)26 were transfected into human BM-MSC with Lipofectamine™ RNAiMAX reagent (Life Technologies). Cells were analyzed 48 h after transfection.
Viral vectors and cell transduction
Retroviral overexpression of GATA2 was performed using the MSCV retrovirus vector, which co-expresses green fluorescent protein (GFP) by internal ribosome entry sites (IRES), transfecting into Platinum Retroviral Packaging Cell Lines (PLAT-F)27 with FuGENE HD (Roche). Human BM-MSC were pretreated with Retronectin (TAKARA BIO.), and GFP-positive cells were sorted using FACSAria II (BD Biosciences).
Co-culture of CD34-positive-enriched cells with a mesenchymal stem cell feeder layer
BM-MSC were transfected with control or GATA2-siRNA. On day 3, control and GATA2 knockdowned BM-MSC, respectively, were replaced with serum-free medium containing CD34-positive-enriched cells (RIKEN). Serum-free medium (StemPro-34 SFM: Life Technologies) contained 100 ng/mL stem cell factor, 100 ng/mL interleukin (IL)-3, and 25 ng/mL granulocyte-monocyte colony-stimulating factor (Peprotech). The cells were co-cultured for 7 days, and subsequently harvested and analyzed with FACSAria II (BD).
Colony-forming cell assay
CD34 positive-enriched cells, co-cultured with BM-MSC for 7 days, were seeded into semisolid culture (MethoCult™ H4435, Stem Cell Technologies). After 14 days, colony-forming units were counted.
Cell proliferation and cell cycle analysis
The total number of viable cells was determined by a colorimetric method using MTS (3–4,5-dimethylthiazol-2-yl-5-3-carboxymethoxyphenyl-2-4-sulfophenyl-2H-tetrazolium, inner salt; CellTiter 96). Absorbance at 490 nm was measured with an iMark microplate reader (Bio-rad). For cell cycle analysis, cells were fixed in ice-cold 70% ethanol and stained with 20 μg/mL propidium iodide (Sigma), 0.2 mg/mL RNase (Sigma), and 0.1% Triton X-100 (Sigma). DNA content was determined using FACSAria II and FlowJo software (http://www.flowjo.com/).
Statistical significance was assessed using a two-sided Student t-test.
Acceleration of adipocyte differentiation in mesenchymal stem cells from GATA2 knockout mice
We first generated BM-MSC from bone marrow cells of conditional GATA2 knockout mice (GATA2), in which the DNA binding domain of GATA2 (exon 5 encoding the C-terminal zinc-finger motif) could be deleted by inducing the Cre-loxP system (GATA2−) (Online Supplementary Figure S1A). We confirmed that GATA2 BM-MSC retained the potential to differentiate into adipogenic lineages (Online Supplementary Figure S1B). Flow cytometric analysis confirmed the characteristic immunophenotype,28 showing that GATA2 BM-MSC expressed CD29, CD44 and Sca-1 but not markers such as CD11b, CD34 and CD45 (Online Supplementary Figure S1C).
To determine whether the loss of GATA2 influenced the BM-MSC phenotype, the DNA-binding domain of GATA2 was deleted using the Cre-loxP system, and GATA2 knockout–BM-MSC (GATA2 BM-MSC) were generated. Quantitative RT-PCR analysis revealed that Gata2 expression was significantly decreased in the GATA2 MSC, implying that iCre-mediated deletion of the GATA2 C-finger resulted in decreased GATA2 autoregulation (Figure 1A). When GATA2 and GATA2− BM-MSC were exposed to adipogenic differentiation stimuli, we observed an overall increase in the expression of Cebpa (CEBPα), Pparg (PPARγ), and Fabp4 (aP2) in GATA2 BM-MSC (Figure 1B). Moreover, the expression of these genes peaked during days 8–12 of differentiation and then dropped to levels similar to those of control cells (Figure 1B), whereas the expression level of Cebpb (CEBPβ) was slightly higher in GATA2 BM-MSC at the early (day 4) and last (day 16) stages of differentiation (Figure 1B). Furthermore, oil drop formation was markedly increased in GATA2 MSC (Figure 1C). These results suggest that loss of GATA2 function induces the expression of adipogenic factors and adipocyte differentiation of BM-MSC.
Generation and characterization of human bone marrow mesenchymal stem cells
Next, to elucidate the role of GATA2 in the context of human BM-MSC differentiation, we generated BM-MSC from human mononuclear cells derived from bone marrow samples. We confirmed that BM-MSC differentiated into the adipogenic lineage (Online Supplementary Figure S2A). Flow cytometric analysis further confirmed the characteristic immunophenotype,302924 showing that the BM-MSC expressed CD29, CD44, CD90 and CD105 but not CD14, CD34, and CD45 (Online Supplementary Figure S2B).
Short interfering RNA-mediated GATA2 knockdown promotes differentiation of human bone marrow mesenchymal stem cells into adipocytes
To determine whether GATA2 regulates adipocyte differentiation in human BM-MSC, we suppressed GATA2 expression using a specific siRNA. Control or GATA2-siRNA were transfected into human BM-MSC 48 h before inducing adipocyte differentiation. We demonstrated that GATA2 mRNA levels were significantly decreased on day 0 and during adipocyte differentiation until day 8 (Figure 2A B). Thereafter, we analyzed the expression of key adipocyte-specific genes at various time-points during adipocyte differentiation. The levels of expression of C/EBPα, PPARγ and aP2 were significantly increased in the GATA2-knockdowned cells (Figure 2B). Furthermore, oil drop formation on day 12 was significantly increased in the GATA2 knockdown cells, as determined based on the Oil Red O staining-positive area (Figure 2C–D). These findings were consistent with the results for GATA2-deficient murine BM-MSC (Figure 1).
GATA2 overexpression suppresses differentiation of human bone marrow mesenchymal stem cells into adipocytes
We overexpressed GATA2 in human BM-MSC using MSCV-GFP-IRES. After transfecting GATA2-expressing or control retroviruses, GFP-positive cells were sorted. Quantitative RT-PCR assay confirmed GATA2 overexpression (Figure 3A, B). When these cells were differentiated into adipocytes, the levels of expression of C/EBPα, PPARγ, aP2 and Adipsin were significantly diminished by GATA2 overexpression (Figure 3B). Concomitantly, oil drop formation on day 12 was also significantly decreased in cells overexpressing GATA2 (Figure 3C,D).
Taken together, our data suggest that decreased GATA2 expression by human BM-MSC accelerates adipocyte differentiation, whereas GATA2 overexpression suppresses adipocyte differentiation.
Enrichment of cell cycle regulatory genes based on transcriptional profiling to identify GATA2-regulated genes in human bone marrow mesenchymal stem cells
To identify GATA2-target genes in BM-MSC, we conducted comprehensive expression profiling of BM-MSC transfected with control or GATA2-siRNA. Inhibition of GATA2 expression was confirmed based on the profiling data as well as quantitative RT-PCR analysis (0.000104 ± 0.000008 and 0.000198 ± 0.000022, for GATA2 siRNA and control siRNA, respectively, P<0.05) (Table 1, Online Supplementary Table S1, Online Supplementary Figure S3). Based on the average of two independent datasets, we demonstrated that GATA2 knockdown activated and repressed 90 and 189 genes (> 2-fold), respectively (Table 1, Online Supplementary Table S1). The analysis revealed the differential expression of cell-cycle regulators (CHEK1, CCNB1, CCNB2, GTSE1, and CDC20), adhesion molecules (LAMP1 and CD44), as well as ENPP1, which regulate osteoblastic differentiation (Table 1).31 In contrast, and unexpectedly, adipocyte-related genes were not detected. Gene ontology analysis revealed significant enrichment of genes related to “cell cycle” (P=8.6×10) and “protein modification” (P=2.6 ×10; Table 2).
As described above, we identified decreased expression of various cell cycle regulatory genes after GATA2 knockdown. Previous studies of hematopoietic cells have suggested that GATA2 expression varies during the cell cycle and that GATA2 regulates cell-cycle regulators.3332 We, therefore, evaluated whether the cell cycle was altered in BM-MSC in which GATA2 expression was inhibited. The number of cells present in G1/G0 was significantly increased when GATA2 expression was decreased (Figure 4), which was due to the significantly increased proportion of cells in the G1 phase (Online Supplementary Figure S4). We further confirmed that cell proliferation was decreased by decreased GATA2 expression (Figure 5). These results suggest that GATA2 has an important role in BM-MSC proliferation by regulating cell-cycle regulators.
Reduced hematopoietic support of human bone marrow mesenchymal stem cells by GATA2 knockdown
Although BM-MSC differentiate into various cell types that form the hematopoietic microenvironment, BM-MSC themselves can support HSC.34 To determine whether the ability of BM-MSC to support HSC was compromised by decreasing GATA2 expression levels, we co-cultured cord blood-derived CD34-positive cells with BM-MSC that were transfected with control or GATA2 siRNA. After co-culture of CD34-positive cells with the BM-MSC, the HSC fraction was isolated using the gating strategy of the International Society of Hematotherapy and Graft Engineering (ISHAGE) (Figure 6A).3635 The frequency of CD34-positive cells on day 7 tended to decrease upon co-culturing with BM-MSC with GATA2 knockdown, but this was not statistically significant (Figure 6B). Subsequently, we assessed the colony-forming capacity of CD34-positive cells after culture with each siRNA-treated BM-MSC. As shown in Figure 6C–E, the total number of colonies was significantly lower when the CD34-positive cells were cultured with BM-MSC transfected with the GATA2-siRNA. To compare the effects with a more advanced stage of adipocyte differentiation, we conducted the same series of analyses based on BM-MSC-derived adipocytes, demonstrating that HSC frequency and colony formation were decreased by GATA2 knockdown (Figure 7). In brief, our data suggest that the decrease of GATA2 expression in BM-MSC decreased the cells’ ability to support the hematopoietic microenvironment.
As noted, GATA2 expression in CD34-positive cells is significantly decreased in patients with aplastic anemia by an unknown mechanism.2019 Because cytokines such as transforming growth factor-β,37 interferon-γ,37 tumor necrosis factor-α,3938 IL-6,39 IL-17A,39 and IL-1β4140 may be involved in the pathogenesis of aplastic anemia, we evaluated whether the addition of these cytokines may accelerate adipocyte differentiation. Unexpectedly, adipocyte differentiation was suppressed by these cytokines, except for IL-6 (Online Supplementary Figure S5A–F). In addition, the suppression of adipocyte differentiation did not always correlate with the changes of GATA2 expression level, nor AP2 expression (i.e. tumor necrosis factor-α and IL-6), possibly because these cytokines might affect AP2 expression level and adipocyte differentiation independently of GATA2. Next, we assessed the effect of bone morphogenic protein (BMP)-4, because a previous study demonstrated that BMP4 regulates GATA2 expression in embryonic stem cells.42 As shown in Online Supplementary Figure S5G, BMP4 suppressed adipocyte differentiation and significantly induced GATA2, suggesting that BMP4 could be one of the factors involved in the regulation of GATA2 in BM-MSC.
The balance between proliferation and differentiation of MSC may be tightly regulated by BMP, Hedgehog, and Wnt signaling pathways, among others.54433 BMP2 and BMP4 promote adipocyte differentiation.47 Noggin inhibits BMP signaling and promotes osteogenic differentiation.43 In addition, Hedgehog and Wnt signaling pathways inhibit adipocyte differentiation but promote osteoblastic differentiation.4844 Efforts to identify the regulatory mechanisms that control the differentiation of BM-MSC into adipocytes/osteoblasts may lead to the development of new clinical applications in the fields of regenerative medicine and tissue engineering and enhance our understanding of hematopoiesis, since BM-MSC are the primary sources of the hematopoietic microenvironment.1
Although GATA2 may play an important role in regulating adipocyte differentiation from a mouse preadipocytic cell line,1816 its role in regulating human BM-MSC differentiation is unknown. In the present study, we revealed that knockdown and overexpression of GATA2 accelerated and inhibited adipocyte differentiation, respectively (Figures 2 and 3). We assumed that BM-MSC fate might be determined by the balances of multiple transcription factors, rather than solely by GATA2. For example, GATA1 and PU.1, master regulators in erythroid and granulocyte differentiation, respectively, act mutually antagonistically.5655 Similar antagonism has also been reported between C/EBPα and PU.1 during neutrophil differentiation.5857 Furthermore, in murine MSC, the propensity for differentiation toward osteoblasts or adipocytes was affected by various factors including Maf, Runx2, Cebpb and Pparg.6059 Nevertheless, our data clearly demonstrate that GATA2 could be one of the important factors that determine immaturity and differentiation toward adipocytes in BM-MSC.
We have demonstrated that GATA2 knockdown in BM-MSC increased the number of cells in G0/G1 (Figure 4, Online Supplementary Figure S4), with significant downregulation of cell cycle regulators such as CHEK1, CCNB1, CCNB2, GTSE1, and CDC20 (Table 1). GATA2 expression oscillates during the cell cycle such that expression is high in the S phase but low in G1/S and M phases.32 Moreover, GATA2 regulates cell cycle regulators, including CCND3, CDK4, and CDK6,33 suggesting that GATA2 contributes to the regulation of the HSC pool within the bone marrow. We, therefore, conclude that GATA2-mediated cell cycle regulation occurs in BM-MSC. Recent evidence suggests that adipocyte differentiation is triggered during the G1 phase when progenitor cells are exposed to adipogenic stimuli such as insulin, insulin-like growth factor 1, dexamethasone, and cyclic AMP.6261 Furthermore, considering our finding that GATA2 knockdown in BM-MSC increased the number of G1 phase cells (Figure 4, Online Supplementary Figure S4), the susceptibility to adipogenic stimuli may have been augmented by GATA2 knockdown, leading to accelerated adipocyte differentiation.
We have also demonstrated that GATA2 expression in HSC is lower in patients with aplastic anemia and leads to decreased HOXB4 expression,2019 which would contribute to the reduction in the size of the HSC pool.26 Furthermore, GATA2 expression is also lower in BM-MSC derived from patients with aplastic anemia.13 In the present study, we demonstrated that decreased GATA2 expression in BM-MSC accelerated adipocyte differentiation. Thus, decreased GATA2 expression by HSC and BM-MSC may lead to decreased number of HSC as well as fatty marrow change, which is a characteristic feature of aplastic anemia.
In addition to the accelerated adipocyte differentiation from BM-MSC by GATA2 knockdown (Figure 2), we further demonstrated that GATA2-knockdowned BM-MSC compromised colony-forming capacity based on co-culture with CD34-positive cells (Figure 6). Although the molecular mechanism responsible for this effect is unknown, decreased expression of cell adhesion molecules such as LAMB1, CD44, and FBN2 (Table 1) may be involved in impaired HSC maintenance. LAMB1 encodes laminin β1, which is expressed in the hematopoietic microenvironment, and contributes to the regulation of hematopoiesis.63 Furthermore, CD44 promotes the homing process between HSC and the hematopoietic niche through hyaluronic acid, which serves as a ligand.6564 Thus, GATA2 downregulation in patients with aplastic anemia may result in a decrease of extracellular matrix, similar to the functions of LAMB1 and CD44, resulting in impaired HSC support.
Immunosuppressive therapy is effective in 75% of cases of aplastic anemia, suggesting that immunological injury plays a role in the pathogenesis of aplastic anemia.66 However, in our study, the addition of various cytokines did not accelerate adipocyte differentiation or decrease GATA2 expression (Online Supplementary Figure S5), suggesting that transforming growth factor-β, interferon-γ, tumor necrosis factor-α, IL-6 and IL-17A and IL-1β might not be involved in the regulation of GATA2 expression in BM-MSC. We included BMP4 in our analysis because this protein regulates GATA2 expression.42 As expected, we demonstrated that BMP4 induced GATA2 expression, suggesting that BMP4 may affect GATA2 expression in BM-MSC (Online Supplementary Figure S5G). However, we observed that the addition of BMP4 suppressed adipocyte differentiation, unlike the results of another study on preadipocytic C3H10T1/2 cell lines.47 We suggest that this difference may be attributed, in part, to the concentration of BMP4 used here. Nevertheless, in addition to BMP signaling, several factors, such as the Wnt signaling pathway, regulate GATA2 expression.67 Further analyses are, therefore, required to elucidate the pathogenesis of aplastic anemia.
In conclusion, our findings support the hypothesis that GATA2 plays an important role in regulating the differentiation potential of BM-MSC and contributes to hematopoietic supporting capacity. Therefore, in bone marrow, GATA2 is not only involved in the generation and/or maintenance of HSC, but also in regulating the hematopoietic microenvironment. Identifying the regulatory mechanism of GATA2 in HSC and BM-MSC may lead to the development of novel therapeutic approaches for bone marrow failure syndromes.
We thank the staff of the Department of Hematology and Rheumatology for helpful discussions and members of the Biomedical Research Core of Tohoku University School of Medicine and Biomedical Research Unit of Tohoku University Hospital for support. We thank Drs. T Kitamura, M Kurokawa and S Camper for providing the PLAT-F retroviral vector, iCre-producing MP34 cell line and GATA2 conditional knockout mice, respectively. All animal experiments were conducted at the Institute for Animal Experimentation, Tohoku University Graduate School of Medicine.
- The online version of this article has a Supplementary Appendix.
- Authorship and Disclosures Information on authorship, contributions, and financial & other disclosures was provided by the authors and is available with the online version of this article at www.haematologica.org.
- Received February 12, 2014.
- Accepted August 7, 2014.
- Frenette PS, Pinho S, Lucas D, Scheiermann C. Mesenchymal stem cell: keystone of the hematopoietic stem cell niche and a stepping-stone for regenerative medicine. Annu Rev Immunol. 2013; 31:285-316. PubMedhttps://doi.org/10.1146/annurev-immunol-032712-095919Google Scholar
- Naveiras O, Nardi V, Wenzel PL, Hauschka PV, Fahey F, Daley GQ. Bone-marrow adipocytes as negative regulators of the haematopoietic microenvironment. Nature. 2009; 460(7252):259-63. PubMedhttps://doi.org/10.1038/nature08099Google Scholar
- Nishikawa M, Ozawa K, Tojo A, Yoshikubo T, Okano A, Tani K. Changes in hematopoiesis-supporting ability of C3H10T1/2mouse embryo fibroblasts during differentiation. Blood. 1993; 81(5):1184-92. PubMedGoogle Scholar
- Calvi LM, Adams GB, Weibrecht KW, Weber JM, Olson DP, Knight MC. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature. 2003; 425(6960):841-6. PubMedhttps://doi.org/10.1038/nature02040Google Scholar
- Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD. Multilineage potential of adult human mesenchymal stem cells. Science. 1999; 284(5411):143-7. PubMedhttps://doi.org/10.1126/science.284.5411.143Google Scholar
- Lowell BB. PPARgamma: an essential regulator of adipogenesis and modulator of fat cell function. Cell. 1999; 99(3):239-42. PubMedhttps://doi.org/10.1016/S0092-8674(00)81654-2Google Scholar
- Tang QQ, Lane MD. Adipogenesis: from stem cell to adipocyte. Annu Rev Biochem. 2012; 81:715-36. PubMedhttps://doi.org/10.1146/annurev-biochem-052110-115718Google Scholar
- Cao Z, Umek RM, McKnight SL. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev. 1991; 5(9):1538-52. PubMedhttps://doi.org/10.1101/gad.5.9.1538Google Scholar
- Rangwala SM, Lazar MA. Transcriptional control of adipogenesis. Annu Rev Nutr. 2000; 20:535-59. PubMedhttps://doi.org/10.1146/annurev.nutr.20.1.535Google Scholar
- Tsai FY, Keller G, Kuo FC, Weiss M, Chen J, Rosenblatt M. An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature. 1994; 371(6494):221-6. PubMedhttps://doi.org/10.1038/371221a0Google Scholar
- Harigae H. GATA transcription factors and hematological diseases. Tohoku J Exp Med. 2006; 210(1):1-9. PubMedhttps://doi.org/10.1620/tjem.210.1Google Scholar
- Bresnick EH, Lee HY, Fujiwara T, Johnson KD, Keles S. GATA switches as developmental drivers. J Biol Chem. 2010; 285(41):31087-93. PubMedhttps://doi.org/10.1074/jbc.R110.159079Google Scholar
- Xu Y, Takahashi Y, Wang Y, Hama A, Nishio N, Muramatsu H. Downregulation of GATA-2 and overexpression of adipogenic gene-PPARgamma in mesenchymal stem cells from patients with aplastic anemia. Exp Hematol. 2009; 37(12):1393-9. PubMedhttps://doi.org/10.1016/j.exphem.2009.09.005Google Scholar
- Tsai FY, Orkin SH. Transcription factor GATA-2 is required for proliferation/survival of early hematopoietic cells and mast cell formation, but not for erythroid and myeloid terminal differentiation. Blood. 1997; 89(10):3636-43. PubMedGoogle Scholar
- Zhou Y, Yamamoto M, Engel JD. GATA2 is required for the generation of V2 interneurons. Development. 2000; 127(17):3829-38. PubMedGoogle Scholar
- Tong Q, Dalgin G, Xu H, Ting CN, Leiden JM, Hotamisligil GS. Function of GATA transcription factors in preadipocyte–adipocyte transition. Science. 2000; 290(5489):134-8. PubMedhttps://doi.org/10.1126/science.290.5489.134Google Scholar
- Okitsu Y, Takahashi S, Minegishi N, Kameoka J, Kaku M, Yamamoto M. Regulation of adipocyte differentiation of bone marrow stromal cells by transcription factor GATA-2. Biochem Biophys Res Commun. 2007; 364(2):383-7. PubMedhttps://doi.org/10.1016/j.bbrc.2007.10.031Google Scholar
- Tong Q, Tsai J, Tan G, Dalgin G, Hotamisligil GS. Interaction between GATA and the C/EBP family of transcription factors is critical in GATA-mediated suppression of adipocyte differentiation. Mol Cell Biol. 2005; 25(2):706-15. PubMedhttps://doi.org/10.1128/MCB.25.2.706-715.2005Google Scholar
- Fujimaki S, Harigae H, Sugawara T, Takasawa N, Sasaki T, Kaku M. Decreased expression of transcription factor GATA-2 in haematopoietic stem cells in patients with aplastic anaemia. Br J Haematol. 2001; 113(1):52-7. PubMedhttps://doi.org/10.1046/j.1365-2141.2001.02736.xGoogle Scholar
- Zeng W, Chen G, Kajigaya S, Nunez O, Charrow A, Billings EM. Gene expression profiling in CD34 cells to identify differences between aplastic anemia patients and healthy volunteers. Blood. 2004; 103(1):325-32. PubMedhttps://doi.org/10.1182/blood-2003-02-0490Google Scholar
- Charles MA, Saunders TL, Wood WM, Owens K, Parlow AF, Camper SA. Pituitary-specific Gata2 knockout: effects on gonadotrope and thyrotrope function. Mol Endocrinol. 2006; 20(6):1366-77. PubMedhttps://doi.org/10.1210/me.2005-0378Google Scholar
- Goyama S, Yamamoto G, Shimabe M, Sato T, Ichikawa M, Ogawa S. Evi-1 is a critical regulator for hematopoietic stem cells and transformed leukemic cells. Cell Stem Cell. 2008; 3(2):207-20. PubMedhttps://doi.org/10.1016/j.stem.2008.06.002Google Scholar
- Solchaga LA, Penick K, Porter JD, Goldberg VM, Caplan AI, Welter JF. FGF-2 enhances the mitotic and chondrogenic potentials of human adult bone marrow-derived mesenchymal stem cells. J Cell Physiol. 2005; 203(2):398-409. PubMedhttps://doi.org/10.1002/jcp.20238Google Scholar
- Auletta JJ, Zale EA, Welter JF, Solchaga LA. Fibroblast growth factor-2 enhances expansion of human bone marrow-derived mesenchymal stromal cells without diminishing their immunosuppressive potential. Stem Cells Int. 2011; 2011:235176. PubMedGoogle Scholar
- Bianchi G, Banfi A, Mastrogiacomo M, Notaro R, Luzzatto L, Cancedda R, Quarto R. Ex vivo enrichment of mesenchymal cell progenitors by fibroblast growth factor 2. Exp Cell Res. 2003; 287(1):98-105. PubMedhttps://doi.org/10.1016/S0014-4827(03)00138-1Google Scholar
- Fujiwara T, Yokoyama H, Okitsu Y, Kamata M, Fukuhara N, Onishi Y. Gene expression profiling identifies HOXB4 as a direct downstream target of GATA-2 in human CD34+ hematopoietic cells. PLoS One. 2012; 7(9):e40959. PubMedhttps://doi.org/10.1371/journal.pone.0040959Google Scholar
- Sekine R, Kitamura T, Tsuji T, Tojo A. Efficient retroviral transduction of human Blymphoid and myeloid progenitors: marked inhibition of their growth by the Pax5 transgene. Int J Hematol. 2008; 87(4):351-62. PubMedhttps://doi.org/10.1007/s12185-008-0082-7Google Scholar
- Sung JH, Yang HM, Park JB, Choi GS, Joh JW, Kwon CH. Isolation and characterization of mouse mesenchymal stem cells. Transplant Proc. 2008; 40(8):2649-54. PubMedhttps://doi.org/10.1016/j.transproceed.2008.08.009Google Scholar
- Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD. Multilineage potential of adult human mesenchymal stem cells. Science. 1999; 284(5411):143-7. PubMedhttps://doi.org/10.1126/science.284.5411.143Google Scholar
- Boiret N, Rapatel C, Veyrat-Masson R, Guillouard L, Guérin JJ, Pigeon P. Characterization of nonexpanded mesenchymal progenitor cells from normal adult human bone marrow. Exp Hematol. 2005; 33(2):219-25. PubMedhttps://doi.org/10.1016/j.exphem.2004.11.001Google Scholar
- Nam HK, Liu J, Li Y, Kragor A, Hatch NE. Ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) protein regulates osteoblast differentiation. J Biol Chem. 2011; 286(45):39059-71. PubMedhttps://doi.org/10.1074/jbc.M111.221689Google Scholar
- Koga S, Yamaguchi N, Abe T, Minegishi M, Tsuchiya S, Yamamoto M, Minegishi N. Cell-cycle-dependent oscillation of GATA2 expression in hematopoietic cells. Blood. 2007; 109(10):4200-8. PubMedhttps://doi.org/10.1182/blood-2006-08-044149Google Scholar
- Tipping AJ, Pina C, Castor A, Hong D, Rodrigues NP, Lazzari L. High GATA-2 expression inhibits human hematopoietic stem and progenitor cell function by effects on cell cycle. Blood. 2009; 113(12):2661-72. PubMedhttps://doi.org/10.1182/blood-2008-06-161117Google Scholar
- Ding L, Morrison SJ. Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches. Nature. 2013; 495(7440):231-5. PubMedhttps://doi.org/10.1038/nature11885Google Scholar
- Sutherland DR, Anderson L, Keeney M, Nayar R, Chin-Yee I. The ISHAGE guidelines for CD34+ cell determination by flow cytometry. International Society of Hematotherapy and Graft Engineering. J Hematother. 1996; 5(3):213-26. PubMedhttps://doi.org/10.1089/scd.1.1996.5.213Google Scholar
- Brocklebank AM, Sparrow RL. Enumeration of CD34+ cells in cord blood: a variation on a single-platform flow cytometric method based on the ISHAGE gating strategy. Cytometry. 2001; 46(4):254-61. PubMedhttps://doi.org/10.1002/cyto.1136Google Scholar
- Serio B, Selleri C, Maciejewski JP. Impact of immunogenetic polymorphisms in bone marrow failure syndromes. Mini Rev Med Chem. 2011; 11(6):544-52. PubMedhttps://doi.org/10.2174/138955711795843356Google Scholar
- Feng X, Young NS. Cytokine signature profiles in acquired aplastic anemia and myelodysplastic syndromes. Haematologica. 2011; 96(4):602-6. PubMedhttps://doi.org/10.3324/haematol.2010.030536Google Scholar
- Gu Y, Hu X, Liu C, Qv X, Xu C. Interleukin (IL)-17 promotes macrophages to produce IL-8, IL-6 and tumour necrosis factor-alpha in aplastic anaemia. Br J Haematol. 2008; 142(1):109-14. PubMedhttps://doi.org/10.1111/j.1365-2141.2008.07161.xGoogle Scholar
- Hirayama Y, Kohgo Y, Matsunaga T, Ohi S, Sakamaki S, Niitsu Y. Cytokine mRNA expression of bone marrow stromal cells from patients with aplastic anaemia and myelodysplastic syndrome. Br J Haematol. 1993; 85(4):676-83. PubMedhttps://doi.org/10.1111/j.1365-2141.1993.tb03208.xGoogle Scholar
- Ibáñez A, Río P, Casado JA, Bueren JA, Fernández-Luna JL, Pipaón C. Elevated levels of IL-1beta in Fanconi anaemia group A patients due to a constitutively active phosphoinositide 3-kinase-Akt pathway are capable of promoting tumour cell proliferation. Biochem J. 2009; 422(1):161-70. PubMedhttps://doi.org/10.1042/BJ20082118Google Scholar
- Lugus JJ, Chung YS, Mills JC, Kim SI, Grass J, Kyba M. GATA2 functions at multiple steps in hemangioblast development and differentiation. Development. 2007; 134(2):393-405. PubMedhttps://doi.org/10.1242/dev.02731Google Scholar
- Rifas L. The role of noggin in human mesenchymal stem cell differentiation. J Cell Biochem. 2007; 100(4):824-34. PubMedhttps://doi.org/10.1002/jcb.21132Google Scholar
- Ross SE, Hemati N, Longo KA, Bennett CN, Lucas PC. Inhibition of adipogenesis by Wnt signaling. Science. 2000; 289(5481):950-3. PubMedhttps://doi.org/10.1126/science.289.5481.950Google Scholar
- Bowers RR, Lane MD. Wnt signaling and adipocyte lineage commitment. Cell Cycle. 2008; 7(9):1191-6. PubMedhttps://doi.org/10.4161/cc.7.9.5815Google Scholar
- Bennett CN, Longo KA, Wright WS, Suva LJ, Lane TF, Hankenson KD. Regulation of osteoblastogenesis and bone mass by Wnt10b. Proc Natl Acad Sci USA. 2005; 102(9):3324-9. PubMedhttps://doi.org/10.1073/pnas.0408742102Google Scholar
- Zehentner BK, Leser U, Burtscher H. BMP-2 and sonic hedgehog have contrary effects on adipocyte-like differentiation of C3H10T1/2 cells. DNA Cell Biol. 2000; 19(5):275-81. PubMedhttps://doi.org/10.1089/10445490050021186Google Scholar
- Spinella-Jaegle S, Rawadi G, Kawai S, Gallea S, Faucheu C, Mollat P. Sonic hedgehog increases the commitment of pluripotent mesenchymal cells into the osteoblastic lineage and abolishes adipocytic differentiation. J Cell Sci. 2001; 114(Pt11):2085-94. PubMedGoogle Scholar
- van der Horst G, Farih-Sips H, Löwik CW, Karperien M. Multiple mechanisms are involved in inhibition of osteoblast differentiation by PTHrP and PTH in KS483 cells. J Bone Miner Res. 2005; 20(12):2233-44. PubMedhttps://doi.org/10.1359/JBMR.050821Google Scholar
- Isenmann S, Arthur A, Zannettino AC, Turner JL, Shi S, Glackin CA. TWIST family of basic helix-loop-helix transcription factors mediate human mesenchymal stem cell growth and commitment. Stem Cells. 2009; 27(10):2457-68. PubMedhttps://doi.org/10.1002/stem.181Google Scholar
- Xu Y, Zhou YL, Erickson RL, Macdougald OA, Snead ML. Physical dissection of the CCAAT/enhancer-binding protein alpha in regulating the mouse amelogenin gene. Biochem Biophys Res Commun. 2007; 354(1):56-61. PubMedhttps://doi.org/10.1016/j.bbrc.2006.12.182Google Scholar
- Tang QQ, Lane MD. Activation and centromeric localization of CCAAT/enhancer binding proteins during the mitotic clonal expansion of adipocyte differentiation. Genes Dev. 1999; 13(17):2231-41. PubMedhttps://doi.org/10.1101/gad.13.17.2231Google Scholar
- Korenjak M, Brehm A. E2F-Rb complexes regulating transcription of genes important for differentiation and development. Curr Opin Genet Dev. 2005; 15(5):520-7. PubMedhttps://doi.org/10.1016/j.gde.2005.07.001Google Scholar
- Thomas DM, Carty SA, Piscopo DM, Lee JS, Wang WF, Forrester WC. The retinoblastoma protein acts as a transcriptional coactivator required for osteogenic differentiation. Mol Cell. 2001; 8(2):303-16. PubMedhttps://doi.org/10.1016/S1097-2765(01)00327-6Google Scholar
- Zhang P, Behre G, Pan J, Iwama A, Wara-Aswapati N, Radomska HS. Negative cross-talk between hematopoietic regulators: GATA proteins repress PU.1. Proc Natl Acad Sci USA. 1999; 96(15):8705-10. PubMedhttps://doi.org/10.1073/pnas.96.15.8705Google Scholar
- Nerlov C, Querfurth E, Kulessa H, Graf T. GATA-1 interacts with the myeloid PU.1 transcription factor and represses PU.1-dependent transcription. Blood. 2000; 95(8):2543-51. PubMedGoogle Scholar
- Reddy VA, Iwama A, Iotzova G, Schulz M, Elsasser A, Vangala RK. Granulocyte inducer C/EBPalpha inactivates the myeloid master regulator PU.1: possible role in lineage commitment decisions. Blood. 2002; 100(2):483-90. PubMedhttps://doi.org/10.1182/blood.V100.2.483Google Scholar
- Dahl R, Walsh JC, Lancki D, Laslo P, Iyer SR, Singh H. Regulation of macrophage and neutrophil cell fates by the PU.1:C/EBPalpha ratio and granulocyte colony-stimulating factor. Nat Immunol. 2003; 4(10):1029-36. PubMedhttps://doi.org/10.1038/ni973Google Scholar
- Nishikawa K, Nakashima T, Takeda S, Isogai M, Hamada M, Kimura A. Maf promotes osteoblast differentiation in mice by mediating the age-related switch in mesenchymal cell differentiation. J Clin Invest. 2010; 120(10):3455-65. PubMedhttps://doi.org/10.1172/JCI42528Google Scholar
- Sun H, Kim JK, Mortensen R, Mutyaba LP, Hankenson KD, Krebsbach PH. Osteoblasttargeted suppression of PPARγ increases osteogenesis through activation of mTOR signaling. Stem Cells. 2013; 31(10):2183-92. PubMedhttps://doi.org/10.1002/stem.1455Google Scholar
- Lee Y, Bae EJ. Inhibition of mitotic clonal expansion mediates fisetin-exerted prevention of adipocyte differentiation in 3T3-L1 cells. Arch Pharm Res. 2013; 36(11):1377-84. PubMedhttps://doi.org/10.1007/s12272-013-0226-zGoogle Scholar
- MacDougald OA, Lane MD. Transcriptional regulation of gene expression during adipocyte differentiation. Annu Rev Biochem. 1995; 64:345-73. PubMedhttps://doi.org/10.1146/annurev.bi.64.070195.002021Google Scholar
- Siler U, Seiffert M, Puch S, Richards A, Torok-Storb B, Müller CA. Characterization and functional analysis of laminin isoforms in human bone marrow. Blood. 2000; 96(13):4194-203. PubMedGoogle Scholar
- Avigdor A, Goichberg P, Shivtiel S, Dar A, Peled A, Samira S. CD44 and hyaluronic acid cooperate with SDF-1 in the trafficking of human CD34+ stem/progenitor cells to bone marrow. Blood. 2004; 103(8):2981-9. PubMedhttps://doi.org/10.1182/blood-2003-10-3611Google Scholar
- Malfuson JV, Boutin L, Clay D, Thépenier C, Desterke C, Torossian F. SP/drug efflux functionality of hematopoietic progenitors is controlled by mesenchymal niche through VLA-4/CD44 axis. Leukemia. 2013; 28(4):853-64. PubMedGoogle Scholar
- Young NS, Scheinberg P, Calado RT. Aplastic anemia. Curr Opin Hematol. 2008; 15(3):162-8. PubMedhttps://doi.org/10.1097/MOH.0b013e3282fa7470Google Scholar
- Trompouki E, Bowman TV, Lawton LN, Fan ZP, Wu DC, DiBiase A. Lineage regulators direct BMP and Wnt pathways to cellspecific programs during differentiation and regeneration. Cell. 2011; 147(3):577-89. PubMedhttps://doi.org/10.1016/j.cell.2011.09.044Google Scholar