Cell Metabolism

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Production of Functional Classical Brown Adipocytes from Human Pluripotent Stem Cells using Specific Hemopoietin Cocktail without Gene Transfer
Miwako Nishio,1 Takeshi Yoneshiro,4 Masako Nakahara,1 Shinnosuke Suzuki,1 Koichi Saeki,5 Mamoru Hasegawa,5 Yuko Kawai,6 Hidenori Akutsu,7 Akihiro Umezawa,7 Kazuki Yasuda,2 Kazuyuki Tobe,8 Akira Yuo,1 Kazuo Kubota,3 Masayuki Saito,9 and Kumiko Saeki1,*
of Disease Control, Research Institute of Metabolic Disorder, Diabetes Research Center, Research Institute 3Department of Radiology National Center for Global Health and Medicine, Tokyo 162-8655, Japan 4Laboratory of Histology and Cytology, Department of Anatomy, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan 5DNAVEC Corporation, Ibaraki 300-2511, Japan 6LSI Sapporo Clinic, Sapporo 065-0013, Japan 7Department of Reproductive Biology, Center for Regenerative Medicine, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan 8The First Department of Internal Medicine, Faculty of Medicine, University of Toyama, Toyama 930-0194, Japan 9Department of Nutrition, School of Nursing and Nutrition, Tenshi College, Sapporo 065-0013, Japan *Correspondence: saeki@ri.ncgm.go.jp http://dx.doi.org/10.1016/j.cmet.2012.08.001
2Department 1Department

SUMMARY

Brown adipose tissue is attracting much attention due to its antiobestic effects; however, its development and involvement in metabolic improvement remain elusive. Here we established a method for a high-efficiency (>90%) differentiation of human pluripotent stem cells (hPSCs) into functional classical brown adipocytes (BAs) using specific hemopoietin cocktail (HC) without exogenous gene transfer. BAs were not generated without HC, and lack of a component of HC induced white adipocyte (WA) marker expressions. hPSC-derived BA (hPSCdBA) showed respiratory and thermogenic activation by b-adrenergic receptor (AdrRb) stimuli and augmented lipid and glucose tolerance, whereas human multipotent stromal cell-derived WA (hMSCdWA) improved lipid but inhibited glucose metabolism. Cotransplantation of hPSCdBA normalized hMSCdWA-induced glucose intolerance. Surprisingly, hPSCdBAs expressed various hemopoietin genes, serving as stroma for myeloid progenitors. Moreover, AdrRb stimuli enhanced recovery from chemotherapy-induced myelosuppression. Our study enhances our understanding of BA, identifying roles in metabolic and hemogenic regulation.
INTRODUCTION Brown adipose tissue (BAT) is involved in nonshivering thermo¨ ck et al., 1997) and genesis during cold exposure (Enerba diet-induced thermogenesis (Feldmann et al., 2009). It also

contributes to the prevention of aging-associated obesity, as demonstrated in Ucp1 null mice (Kontani et al., 2005). In large-sized mammals, the majority of BAT disappears within a few days after birth; however, some portions remain and function through adulthood. 18Fluorodeoxyglucose-positron emission tomography in combination with computed tomography (18F-FDG-PET/CT) along with histological and gene expressional studies has shown the presence of functional BAT in adult humans in supraclavicular and paravertebral regions (Cypess et al., 2009; Virtanen et al., 2009; van Marken Lichtenbelt et al., 2009; Saito et al., 2009; Yoneshiro et al., 2011). Although accumulating evidence has shown an inverse correlation between the amounts of active BAT and the development of metabolic syndrome in humans (Ouellet et al., 2011; Jacene et al., 2011), the cause-and-effect relationship between BAT and metabolic improvement remains unsubstantiated. Moreover, the whole picture of the development of human BAT is not clarified yet; for example, it remains elusive whether BAT derives from a common progenitor with myoblast, immature mesenchymal cells from which white adipocyte (WA) is also generated or from vascular components such as endothelial and perivascular cells (Tran et al., 2012; Gupta et al., 2012), and whether bone morphogenic protein 7 (BMP7) (Tseng et al., 2008) is sufficient or additional cytokines are required for BA differentiation. The newly proposed concept of brite adipocytes, WA-derived BAlike cells (brown + white = brite) (Petrovic et al., 2010), makes the situation complex, often encumbering an understanding of classical BAT development. For an advanced understanding of BAT, establishing a method to generate BAs from pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), is of great use. Recently, a trial to program human iPSCs (hiPSCs) into BA via transferring of exogenous genes was reported (Ahfeldt et al., 2012); however, biological effects of the programmed BA on lipid/glucose metabolism remains unevaluated. Moreover, artificially programmed

394 Cell Metabolism 16, 394–406, September 5, 2012 ª2012 Elsevier Inc.

2011). Involvement of hPSC-derived BAs (hPSCdBAs) in metabolic improvement. a commonly used feeder for the hematopoietic differentiation of monkey (Hiroyama et al. that for myeloid progenitor cells (MPCs) remains poorly understood. while others showed its inhibiting effects for hematopoiesis (Ookura et al. augmented the expression of UCP1. a de novo hematopoietic stroma must be generated from hPSCs per se.243. Eventually. Effects of hPSC-Derived BA on Lipid and Glucose Metabolism Because endogenous BAT reportedly reduces blood triglyceride (TG) levels in response to cold stimuli (Bartelt et al. UCP1 protein expression was confirmed by immunostaining studies. right). The controversy may come from. showing that over 95% of the BA differentiated cells expressed UCP1 at mitochondria (Figure 2A). we successfully established a highly efficient BA differentiation method for hESCs and hiPSCs (Supplemental Information). showing the presence of a 32 kDa band in the differentiated cells (Figure 2B). the heterogeneity of bone marrow fat cells including BAT versus white adipocyte tissue (WAT). right column) showed reduced fasting TG levels. 394–406. Treatment with a b-adrenergic receptor. 2008) (Figure S2). Together. Naveiras et al. Lipid staining illustrated the wide distribution of mitochondria within the cytosol. CL316. some of which resided close to lipid droplets (Figure 2C). or hMSCdWA.. Quantitative RT-PCR studies demonstrated the induction of BAT-specific genes of UCP1 and PRDM16. Because the hematopoietic differentiation was achieved under a completely feeder-free condition.Cell Metabolism Brown Adipose Production by Hemopoietin Cocktail cells are not applicable to the investigations on natural developmental pathways of human BA. left).0030. 1992).. Ten-week-old mice were subcutaneously injected with saline. we established a high-efficiency method to produce functional BAs from hPSCs including human ESCs (hESCs) and hiPSCs. 2009). but not WATselective genes... 2006) and human (Takayama et al. Also.. 2001) was further confirmed in vivo by subcutaneous transplantation of hPSCdBAs into mice (Figures 3A and 3B.. Arai and Suda. controversial findings have been reported regarding the effects of ‘‘BM adipocyte’’ on the committed HPCs: some reports showed its capacity to support lymphopoiesis (Gimble et al. 1990) and granulopoiesis (Gimble et al. was also determined (Figure 1E). A functional link between BM fat and hematopoiesis was first reported by Dexter et al. 2006. fms-related tyrosine kinase 3 ligand (FLT3LG). thermogenic potential was evaluated. we examined the effects of transplantation of hPSCdBA on lipid metabolism. 395 . (Dexter et al. a major inducer of UCP1 expression. n = 3) and to hMSCdWA-transplanted mice (p = 0. in contrast to hMSCdWA. and also western blotting. was reported in mice (Krings et al. hESC-derived BA (hESCdBA). 2008). Electron micrographs confirmed the presence of multilocular lipid droplets and abundant mitochondria rich in transverse cristae (Figure 2D and Figure S3A). Compared to immature hPSC-transplanted mice. 2012 ª2012 Elsevier Inc. can differentiate into functional BA (Tseng et al.. After a process of trial and error. in hPSCdBAs (Figures 3A and 3B. Upregulation of OCR was further determined in isoproterenol-treated hPSCdBAs (data not shown). a short-term repopulating hematopoietic progenitor cell (HPC) distinct from hematopoietic stem cell (HSC). as confirmed by oil red O staining (Figure 1B). 2012). its service as a stroma for MPCs. effects of hPSCdBAs on glucose metabolism were evaluated. Moreover.. hPSCdBA-transplanted mice (Figure 4A. Thus.. which showed meager mitochondria (Figure S3C). PRDM16 (see Figure S1 online). whereas no significant changes were observed in the cases of hMSCdWA and immature hPSCs (Figure 3D). murine C3H10T1/2 line. we serendipitously found the existence of BA-like cell clusters surrounding the hematopoietic centers and an induction of BA-selective gene. some of which located in close vicinity to lipid droplets (Figure S3B). Fasting blood glucose levels were significantly lowered in hESCdBAtransplanted mice compared to saline-injected mice (p = 0. 2008) ESCs.0032. a nontumorous infiltration of BAT in human BM was reported in a case of essential hyperthrombocythemia (Thorns et al. interleukin-6 (IL-6).. which were not detected in human multipoint stromal cell-derived WA (hMSCdWA) (Figure 1C). which attenuates with aging and diabetes. was also determined: statistically significant upregulation in oxygen consumption rates (OCRs) was determined in the cases of hPSCdBAs in response to CL316.. Olive oil tolerance tests further confirmed that hPSCdBA transplantation augmented resistance to oral lipid loading (Figure 4B).. 2007) has been intensively studied. RESULTS Directed Differentiation of Human PSC into Functional BA By utilizing a specific hemopoietin cocktail (HC) composed of KIT ligand (KITLG). Responsiveness to a b3-adrenergic receptor-selective agonist.243. BA differentiation was completely abolished by HC depletion even in the presence of BMP7 (Figure 1D). During our research into the feeder-free hematopoietic differentiation of hPSCs. who showed that BM adipocytes with mitochondria-attached multilocular lipid droplets were essential for the maintenance of colony-forming units-spleen (CFU-S). 2007) and B cells (Nagasawa. Respiratory activation was also assessed in vitro: hPSCdBAs showed considerably higher basal and maximum OCRs than hMSCdWA as demonstrated by a standard Mito Stress Test (Figure 3C). Next. Expression of a series of BAT-selective and BAT/WAT-common genes. 1977). Cell Metabolism 16.. Although depletion of BMP7 significantly lowered BA differentiation efficiency as reported by Tseng et al. and vascular endothelial growth factor (VEGF) along with the previously reported BA inducer BMP7 (Tseng et al. 2007. and PRDM16. isoproterenol. and blood glucose levels were measured over time (Figure 4C). First. and the existence of BA in vertebral BM are also shown. a major contributor to thermogenesis. September 5. (Tseng et al. 2008). at least in part. an association between BA development and hemopoiesis has been suggested. these findings support the production of functional BAs from hPSCs.. 2008). Isoproterenol-responsive thermogenic activation (Jackson et al. middle column) and hMSCdWA-transplanted mice (Figure 4A. The differentiated cells exclusively contained multilocular lipid droplets (Figure 1A). We next evaluated the functional maturation of hESC/hiPSCderived BAs. The existence of BAT in bone marrow (BM). Although the microenvironment for HSC (reviewed in Kiel and Morrison.

September 5. The error bars represent average ± standard deviation (SD) (n = 3). 396 Cell Metabolism 16. 394–406. 50 mm. Scale bar. 2012 ª2012 Elsevier Inc.Cell Metabolism Brown Adipose Production by Hemopoietin Cocktail Figure 1. Differentiation of hPSCs into BAs (A) Microscopy of hPSC-derived cells. 40 mm. (B) Oil red O staining (right) with phase contrast microscopy (left). . Scale bar. (C) Expression of PRDM16 and UCP1 determined by RT-PCR (left) or real-time PCR (middle and right).

mitochondria. L. because we did not observe any signs of inflammation. n = 3) (Figure 4E). Similar results were obtained when 6-week-old younger mice were used for the assay (data not shown). (D) EM of hiPSC-derived cells (left) and hESCderived cells (right). confirming that hMSCdWA transplantation deteriorates glucose metabolism. significantly lowering the 30 min blood glucose values (p = 0. 2 mm.0003. immature hPSC.Cell Metabolism Brown Adipose Production by Hemopoietin Cocktail Figure 2. BAT/WAT-common. (E) Expression of BAT-selective. The different effects between hESCdBA and hMSCdWA were not due to the difference in cell survival.010. 2002). n = 3). 394–406. (C) Lipid staining. Around the graft tissue. M. n = 3) but also with saline-injected mice (p = 0. hMSCdWA-transplanted mice showed elevated homeostasis model assessment-insulin resistance (HOMA-IR) values compared not only with hESCdBAtransplanted mice (p = 0. as we clearly detected the existence of transplanted cells (Figure S4A). Scale bar. and WAT-selective genes examined by RT-PCR. n = 3) (Figure 4M). differentiated hPSC. 50 mm. n = 3). Finally.B. We also examined longer-term effects of hPSCdBA transplantation using immunocompromised NOG mice (Ito et al. I. All those findings together indicate that (1) hPSCdBAs improve both lipid and glucose metabolism. n = 3) (Figure 4D). we assessed possible therapeutic effect of hESCdBA on hMSCdWA-induced deterioration of glucose metabolism. Scale bar. Although no significant changes in fasting blood glucose values and HOMA-IR values were observed (Figure 4K and 4L). 5 mm.0055. lipid droplets. while hMSCdWA-transplanted mice exhibited elevated 30 min blood glucose values compared not only with hESCdBA-transplanted mice (p = 0. Surprisingly. In addition. Cell Metabolism 16. indicating that hMSCdWA induces insulin resistance despite its favorable effect on lipid metabolism. Cells with multilocular lipid droplet (Figure 4H) that expressed UCP1 (Figure 4I) and human HLA-A. and it expressed only a low level of lL1B (Figure S4C).. As shown in Figure 4F. 50 mm. WA. (2) hMSCdWA improved lipid but deteriorates glucose metabolism. 397 . (B) Western blotting using an anti-UCP1 as indicated. hMSCdWA did not express tumor necrosis factor a (TNFA).0014. and (3) hPSCdBAs ameliorate adverse effects of hMSCdWA on glucose metabolism. deterioration of glucose metabolism by hMSCdWA transplantation could not be attributed to inflammation. Scale bar. (D) BA differentiation in the presence or absence of HC. hESC-derived differentiated cells were stained by an anti-UCP1 antibody (red) and BODIPY 493/503 (green). September 5. 2012 ª2012 Elsevier Inc. hMSCdWA. Similar results were obtained when 6-week-old younger mice were used for the assay (data not shown). Microscopy (upper) and expressions of PRDM16 (lower left) and UCP-1 (lower right) by real time PCR were shown. Analyses on Protein Expressions and Fine Structures (A) Immunostaining using an anti-UCP1 and antiSOD2 antibody as indicated. Fasting blood glucose levellowering effects of hESCdBA were determined at least for 3 weeks (Figure 4G). D.0034. Scale bar. oral glucose tolerance tests (OGTTs) further demonstrated that hESCdBA transplantation reduced 15 min blood glucose values compared to saline injected (p = 0.C (Figure 4J) were determined by histological analyses. The error bars represent average ± SD (n = 3). microvasculatures were also detected (Figure 4H arrowheads). such as macrophage infiltration (Figure S4B). n = 3) but also with saline-injected mice (p = 0. Moreover. cotransplantation of an equivalent number of hESCdBA ameliorated the deleterious effect of hMSCdWA.0011.

. We further evaluated the detailed role of each hematopoietin. Thus. B. The precedence of myoblastic differentiation was further confirmed by the transient induction of PAX3/7. hMSCdWA. or FLT3LG paradoxically induced the expression of a WAT marker. immature hiPSC (ihiPSC).. right. was upregulated. left. hiPSCdBA. the expression of a paraxial mesoderm marker. .. The absence of any one of the components of HC lowered the quality of BA differentiation. Arrows indicate regions of transplantation. BMPR1adependent signaling is required for the survival of immature sphere-forming progenitor cells. depletion of either KITLG. Moreover. IL6. Similar results were obtained from the case of AKT inhibitor (AKT-i). which are involved in myogenic commit- ment (Figure 5B). right). and the omission of any of the HC components results in WAT lineage commitment. Sun et al. Signaling for BA Differentiation To confirm that our differentiation technique correctly reproduced classical BAT development via myoblastic differentiation (Timmons et al. Thermographic images of mice transplanted with saline. immature hESC (ihESC). arrowheads indicate areas of endogenous murine BAT. from which mature BA will be produced. September 5. our method correctly mimics the classical BAT development but not a brite adipocyte pathway via immature mesenchymal stem cell differentiation. Surprisingly. hESCdBA.. The error bars in (C) and (D) represent average ± SD (n = 3). B.. VEGFR2 (Sakurai et al. NG2 and PDGFRB (Crisan et al. or hiPSCdBA before and after isoproterenol treatments are shown (A. Thus. or FLT3LG was required for subsequent UCP1 expression (Figure 5D). Cell death had been induced as early as day 1 (data not shown). 2011). were reduced. (C) Mito stress tests were performed using hESCdBA. 2006). Gene expression was examined by RT-PCR over time (A. effects of these two inhibitors. the expression of a series of developmental markers was examined. myoblastic MYF5 expression was transiently upregulated during the initial floating culture step of differentiation. 2007. IL6. hESCdBA (A) and hiPSCdBA (B) were treated with isoproterenol. Gene expression studies further showed that VEGF was required for PRDM16 expression.243. 2008. second left). the levels of immature mesenchymal stem cell marker. We found that BMPR1a-i induced massive cell death during the floating culture step of BA differentiation (Figure 5E. Because a BMPR1a inhibitor (BMPR1a-i) and p38 MAPK inhibitor (p38-i) reportedly hamper BA differentiation (Sellayah et al. (D) OCR was measured in hESCdBA. along with those of a MAP kinase-ERK kinase (MEK) inhibitor (MEK-i). the HC is essential for the differentiation of hPSCs into classical BA. 2011). We also examined intracellular signaling by performing inhibitor analyses. were examined. right). Seale et al. and hMSCdWA as indicated. and immature hiPSCs after a 4 hr incubation with or without CL316. vegfr2. (Figure 5E. left). phosphoserine aminotransferase 1 (PSAT1) (Seale et al. Thus. reducing cellular viability and/or percentages of multilocular lipid-containing cells (Figure 5C). We then followed up the p38-i. 2006). immature hESCs. Thermogenic and Mitochondrial Respiratory Activation (A and B) Thermogenesis studies. platelet-derived growth factor receptor a (PDGFRA) (Sakurai et al. and a lateral plate mesoderm marker.. 394–406. as well as a lateral plate mesoderm marker..Cell Metabolism Brown Adipose Production by Hemopoietin Cocktail Figure 3. As shown in Figure 5A.. By contrast. hiPSCdBA.and MEK-i-treated 398 Cell Metabolism 16. 2007). 2012 ª2012 Elsevier Inc. whereas KITLG. 2008).

5-FU-treated mice were at the nadir at day 3. data not shown). whereas human BM-derived fat cells generated by a dexamethasone/insulin treatment reduce colony-forming capacities of HPCs (Ookura et al. Compatible to these morphological findings. Moreover. when a decline in total enucleated cell number (11. murine BM fat reportedly expresses various BA-selective messages (Krings et al. (Hofer et al. 2006) and human (Takayama et al.96 [3106].. By virtue of its technological merits. p = 0. Various hematopoietin genes including thrombopoietin (THPO). Compatibly.93 ± 1. n = 3) (Figure 6F) as well as a reduction in early myeloid cells (Figures S5A–S5C) were observed. is widely used as a feeder for the hematopoietic differentiation of monkey (Hiroyama et al.. and bone marrow cells were collected and analyzed over time (Figure 6E). isoproterenol-treated mice showed higher enucleated cell number (10. We also examined the expression of hematopoietic cytokines involved in the expansion and differentiation of committed HPCs.33 ± 1. UCP1 and PRDM16. As reported by Hofer et al. 2007). DISCUSSION We established a highly efficient method for the differentiation of hPSCs into functional BAs. 2008). Collectively. CD34+ cells were directly transplanted without culturing on hPSCdBA layers (Figure 6A). colony- stimulating factor 2 (CSF2). 1992). expressions of BAT-specific markers. 2011). Moreover. splenic chimerisms were significantly higher in hPSCdBA-cocultured CD34+-transplanted mice than in mice with direct transplantation (p = 0. The existence of BA in BM has long been suggested despite the lack of direct evidence (reviewed in Motyl and Rosen.8 of age. but not MEK-i treatment. whereas MEK-i treatment induced cogeneration of the cells with unilocular lipid droplets (Figure 5F. 1973). were examined using commercially available human BM RNA samples. further supporting the notion that hPSCdBA serves as stromal for committed HPCs. We identified cold-stimulated 18 F-FDG uptake in vertebral BM (Figures 7B–7D). while no significant changes in B lymphocyte percentages were observed (data not shown). middle lanes) and hiPSCdWA (data not shown).. left half). On the other hand.87 ± 0. Mice were treated with 5-fluorouracile (5-FU). 2007).. To evaluate in vivo relevance. Cytological studies confirmed all those findings (Figure S5C). but not WA. colony-stimulating factor 3 (CSF3). the expression levels of the hematopoietin genes in hESCdBA (Figure 6D) and hiPSCdBA (data not shown) were upregulated by isoproterenol treatments..19.. 399 . 2010). As shown in Figure 6B.Cell Metabolism Brown Adipose Production by Hemopoietin Cocktail cells until day 10. human umbilical cord blood CD34+ HPCs were cultured on hESCdBA layers for 1 week in the absence of any recombinant hematopoietic cytokines.. lower middle).0023.032. spleen. (3) BM is replaced by WA in severe myelosuppressive states including aplastic anemia. To validate our hypothesis. Yoneshiro et al. A Functional Link between BA and Hematopoiesis There has been a controversy regarding the effect of BM adipocytes on the proliferation and differentiation of committed HPCs. heart. (2) the murine embryo-derived C3H10T1/2 cell line (Reznikoff et al. which differentiates into mature BAT on BMP7 treatment (Tseng et al. The presence of vertebral BM was histologically examined using 3-week-old murine vertebral BM samples: we successfully detected the cells with BA morphologies (Figure 7E) that were positive for UCP1 protein expression (Figure 7F). splenic chimerisms were measured to assess the expansion of CFU-S. PRDM16 and UCP1 expressions were only slightly reduced in p38-i-treated hiPSCs but clearly reduced in MEK-treated hiPSCs (Figure 5G. In the case of hESC. promoting their expansion/differentiation and homing to the spleen. As shown in Figure 7A. upper middle). n = 20) with or without cold stimuli (Saito et al. our method provides a valuable tool for BAT research. p38-i treatment. n = 3. but not of those of brain. By contrast. the mice still suffered from a shortage of mature myeloid cells (Figures S5A–S5C). p = 0. Then. upper right).74 versus 4. 2008) ESCs. and erythropoietin (EPO) were expressed in hESCdBA (Figure 6C. and (4) treatment with dexamethasone/insulin induces differentiation into WA but not BA. We hypothesized that the controversy came from the heterogeneity of BM adipocytes and that BA. Therefore. 2011). serves as a stroma for committed HPCs for the following reasons: (1) hematopoietic stromal cells essential for maintaining CFU-S exhibit morphological resemblance to BA rather than to WA (Dexter et al. while MEK-i treatment exerted minimal effects (Figure 5F. 18F-FDG-PET/CT examinations were performed in healthy young volunteers (24.8 ± 0. n = 3) (Figure 6F) with significantly larger R2 fraction percentages (Figures S5A and S5B). 2009. 2012). p38-i treatment reduced the number of lipid dropletcontaining cells (Figure 5F. we examined whether isoproterenol treatment could enhance the recovery from antitumor agent-induced myelosuppression by enhancing the expansion/ differentiation of MPCs. or muscle (Figures S6A and S6B). 2012 ª2012 Elsevier Inc.70 ± 0. percentages of human CD33-positive myeloid cells were larger in cocultured CD34+-transplanted mice (4. These findings indicate that hPSCdBA serves as a stroma for MPCs.00022.. those findings strongly suggest the presence of functional BA in the BM of vertebrae. p38 MAPK and ERK signaling play important roles in BA differentiation depending on the lines or kinds of hPSCs.. murine BM adipocytic lines are reportedly capable of supporting lymphopoiesis (Gimble et al. p = 0. A relationship between osteoblast and BA was reported in mice (Calo et al.8 ± 5. floating cells were subjected to intrabone marrow transplantation (IBM-T) into alymphocytic NOG mice. right half). whose signal intensities showed an intimate correlation with those of BATs (p < 0.. p38-i treatment exerted minimal effects on hiPSCs (Figure 5F. September 5.. whereas they were undetectable in human BMoriginated MSC-derived WA (hBM-MSCdWA). To assess the possible existence of BA in human BM.0 ± 0. This is the first success in generating functional classical BA pluripotent stem cells without exogenous gene transfer. IL6. lower right). Functional Cell Metabolism 16. 1977). IL3. and after 8 weeks. when mature BA was generated. Moreover.14. Although total cell number (8.. expression of both genes was detected by RT-PCR. n = 3). 1990) and granulopoiesis (Gimble et al. To further assess the existence of active BM-BAT in vivo. n = 3) (Figure 6F) and the percentages of R1 fraction (Figures S5A and S5B) were eventually upregulated at day 7 as a sign of a recovery from myelosuppression. For example.13 versus 3. By contrast.. 394–406. For a control.001). hMSCdWAs expressed only IL6 among these hemopoietins (Figure 6C.40.041. lowered PRDM16 and UCP1 expression levels (Figure 5G. right lanes).

After 16 hr starvation. hESCdBA (n = 3 mice). September 5. . Fasting blood glucose levels (D). or hMSCdWA (n = 3 mice). fasting blood glucose levels were measured. (B) Oral fat tolerance tests. (C–F) OGTT. or hMSCdWA (n = 3 mice). Metabolic Improvement by hPSC-Derived BA Transplantation (A) Blood TG clearance tests. Histological studies were performed by HE staining (H) and 400 Cell Metabolism 16. At indicated time points. hESCdBA (n = 3 mice). ICR mice were injected with saline (n = 3 mice). and blood TG levels were measured over time after isoproterenol treatments. 394–406. isoproterenol was administrated and blood TG levels were measured. 2012 ª2012 Elsevier Inc.Cell Metabolism Brown Adipose Production by Hemopoietin Cocktail Figure 4. and OGTT was performed (C). (G–J) Immunocompromized NOG mice were injected with saline or transplanted with hESCdBA. HOMA-IR (E). ICR mice were transplanted with immature hiPSC (ihiPSC) (n = 3 mice) or hiPSCdBA (n = 3 mice). and blood glucose values after oral glucose loading (F) are shown. Olive oil was orally loaded. Three mice (day 10) or five mice (Day 21) were used for each condition (G). Immunocompetent ICR mice were transplanted with immature hESCs (ihESC) (n = 3 mice).

1999). which is responsible to a b3-adrenergic receptor agonist. 2008) and because Myf5-positive cells emerge at the juxtaspinal. WAKO Pure Chemical Industries). which are capable of unlimited expansion in vitro. 1976).. Among those. Further investigation will elucidate the whole picture of HPC regulation. Transgenes were eliminated by a 395C heat treatment for 5 days. reflecting the individual difference of the ‘‘donor’’ or the difference in the type of pluripotent stem cells or both. the ‘‘stroma’’ for committed HPCs remains a mystery. Our system. IL6. in BA differentiation of hESC (Figures 5E–5G). we demonstrated the differential effects on metabolic regulation between BA and WA: BA improves while WA deteriorates glucose metabolism. (Dexter et al. Our findings imply that VEGF and IL6. the major side effect of intensive chemotherapy for progressive cancers. Because those effects were confirmed by a short-term assay without body weight changes and also because the effects of BA and WA on lipid metabolism were similar... Scale bars. Inc. At this moment. In contrast to the ‘‘niche’’ for HSCs. (K). the beneficial effect of BA on glucose metabolism is not a secondary consequence of general metabolic improvement. Inc. WAKO Pure Chemical Industries).monothioglycerol (207-09232. 394–406. prospectively paravertebral. regions within somites (Cossu et al. One of the main findings of our research is that HC composed of KITLG.). 450 mM a. 5 ng/ml VEGFA. 2008). Because the major portions of vertebrae are composed of trabecular bones. Life Technologies. work as fundamental autocrine or paracrine factors to promote BA differentiation. 20 ng/ml BMP4. Tonello et al. (K–M) Mice were transplanted with hMSCdWAs alone or together with hESCdBA. 50 mm. Arrowheads in (H) indicate microvasculatures. strongly suggest the existence of active BAT in vertebral BM in mammals. 401 . and OGTT was performed. The immunostaining using an anti-UCP1 antibody (I) and anti-human HLA-A. Life Technologies. For clinical application. IL6 is reportedly secreted from cultured human BM adipocytes (Laharrague et al. and blood glucose values after oral glucose loading (M) are shown. 2. Life Technologies. 50 mg/ml ascorbic acid-2-phosphate (Sigma..). Thus. Another surprising finding is the functional link between BA and hematopoiesis. Compatible to the finding by Sellayah et al. which is composed of immature osteoblasts and sinusoidal endothelial cells. EXPERIMENTAL PROCEDURES Establishment of hiPSCs and Provision of hESCs SeV-iPS cells were established from human neonatal fibroblast or human umbilical vein endothelial cells by introducing Yamanaka’s four factors using CytoTune-iPS ver.5 ng/ml IL-6) for 8 days to form spheres. (L).. we have found that BA differentiation efficiencies substantially differ among hiPSC lines (data not shown). 2008).5 ng/ml FLT3LG. but not of MEK signaling. Braun and Arnold. and (3) b-adrenergic receptor stimuli accelerate the recovery from 5-FU-mediated myelosuppression together show that BM-BAT serves as a stroma for MPCs. 2000). 1996. (2) hPSCdBAs promote myelopoiesis of human cord blood CD34+ cells. Fasting blood glucose levels (K).). distinct findings were obtained from the case of hiPSCs. 1996). the hematopoietic microenvironment of vertebral BM may bear a unique character.Cell Metabolism Brown Adipose Production by Hemopoietin Cocktail BA. Further investigations are required to obtain the whole picture of the molecular basis for BA differentiation of hPSCs. 5 mg/ml bovine serum albumin (A802. 1:100 insulin-transferrin-selenium (ITS-A. However. Sigma Chemical Co.. Although BMP7 plays an important role in BA differentiation of hPSC (Figure S2) as reported in murine cases (Tseng et al. in which BM fat cells with multilocular lipid droplets attached by mitochondria were identified as a stroma for CFU-S. which are the sites of active hematopoiesis. 2006). The merit of our system is that it does not require preparation of human specimen materials but utilizes hPSCs. (Sellayah et al. HOMA-IR (L). A hESC line (KhES-3) was generously provided by the Institute for Frontier Medical Science. The error bars in (A). We showed that hPSCdBAs serve as a stroma for MPCs. It may be related to the difference in the genetic background of pluripotent stem cell lines. the third finding is particularly important because it illustrates a very feasible way to shorten the period of myelosuppression. A-8960). Our results indicating that (1) hPSCdBAs express various hematopoietic cytokines in response to b-adrenergic receptor stimuli. By providing high-purity human BA and WA materials. our system will provide a unique tool for the research of BA in regard to glucose metabolism. Inc. Conventional subcutaneous fat transplantation experiments were not able to distinguish the effect of BA from that of WA. 2012 ª2012 Elsevier Inc. the inhibitor analyses demonstrated the involvement of p38 MAPK signaling.. providing highly functional hiPSCdBA. in which MEK signaling played a role in BA differentiation (Figure 5E–5G). FLT3LG.1. 2009). the basis for the difference in the effects of identical inhibitors between hESCs and hiPSCs remains elusive. 5% protein-free hybridoma mix (PFHMII.).. Moreover. Inc.. The PET-CT results of young healthy volunteers. GIBCO #12040-077. The only report showing the characteristic of such stroma was a study by Dexter et al.B.. September 5. 1997. 2.) and Ham’s F12 (087-08335. together with gene expression analyses of human BM specimen and histological examinations of murine vertebral BM samples. HC is indispensable for BA differentiation of hPSCs (Figure 1D). promoting the angiogenesis within BAT. 1:100 synthetic lipids (GIBCO #11905-031. (D)–(G). the existence of BA in vertebral BM seems reasonable.0 (DNAVEC Corp) (Figure S7). Cell Metabolism 16. A Directed Differentiation of hESCs/hiPSCs into Functional BA hESCs or hiPSCs were cultured in a 6 cm low-attachment culture dish using a serum-free differentiation medium composed of 1:1 ratio of IMDM (I3390. Kyoto University (Suemori et al. (B). Because classical BAT is derived from Myf5-positive myoblastic cells (Seale et al. 20 ng/ml KITLG. Sigma Chemical Co. selection of appropriate lines of hiPSCs will be as important as sophisticating the whole differentiation process into good manufacturing practice levels. has also been generated from human multipotent adiposederived stem cells by chronic treatment with thiazolidinediones (Elabd et al. Life Technologies.). may open a new avenue to the therapy for obesity. and the vertebral marrow is the last reserve site for hematopoietic activity in aged individuals (Tanaka and Inoue.. However. 1977). together with KITLG and FLT3LG. because subcutaneous fat tissues contain both WA and BA. 2 mM Glutamax II (GIBCO #35050-061. 2011).C antibody (J) at day 7. and (M) represent average ± SD. as reported in the case of pancreatic b cell differentiation of hESCs (Osafune et al. and VEGF is essential for BA differentiation of hPSCs. It is known that VEGF is synthesized in rodent BAT (Asano et al. and the hematopoietic cytokine cocktail I (5 ng/ml IGF-II.

100 mm (upper panels). Scale bar. (B) Myoblastic marker expressions were determined by RT-PCR during floating culture. Similar results were obtained regarding hiPSCs (data not shown). p38 MAPK. Phase contrast micrographs of the spheres at day 8 (scale bar. or AKT as indicated. 394–406. Expressions of UCP1 and PRDM16 were determined at day 10 by RT-PCR (G). BA differentiation was performed in the presence of inhibitors of BMPR1a. MEK1. 2012 ª2012 Elsevier Inc. 150 mm (lower panels). 200 mm) (E) and those of BA at day 10 (scale bar. Signals Involved in BA Differentiation (A) Developmental marker expression was examined by RT-PCR during BA differentiation of hESCs. (C and D) The role of each cytokine was evaluated by morphological examinations (C) and RT-PCR (D). . and scale bar. 402 Cell Metabolism 16. (E–G) Inhibitor analyses.Cell Metabolism Brown Adipose Production by Hemopoietin Cocktail Figure 5. 50 mm) (F) were shown. September 5.

). Hematopoietin expression in hESCdBAs after isoproterenol treatments was examined over time by RT-PCR (D).5 ng/ml IL-6) for several days. 10 mM BMPR1a inhibitor (Dorsomorphin Dihydrochloride. Germany).. hESCdBAs. Seattle. Inc. WA) as described previously (Nakahara et al. (C and D) Various hematopoietin expression was examined by RT-PCR in immature hESCs (ihESCs). USA) as described in the Supplemental Experimental Procedures. Similar results were obtained at 6 and 12 weeks after transplantation (data not shown). The results were normalized by GAPDH. 2. cells were collected from the spleen and subjected to flow cytometry. Cambridge. UK) as described previously (Nakahara et al. Ltd.. Japan). Osaka. (B) After 8 weeks from transplantation. 2.) or a rabbit polyclonal anti-human SOD2 antibody LS-C39331. The error bars represent average ± SD (n = 3). Inhibitor Analyses BA differentiation was performed by adding the following inhibitors to the differentiation medium: 10 mM p38 MAP kinase inhibitor (Cat 506126) (Calbiochem Co. Quantitative RT-PCR (qPCR) was performed by applying SYBR Green qPCR method using primers purchased from SuperArray (QIAGEN Science. Gene Expression Analyses RT-PCR was performed using primers described in Supplemental Information. Darmstadt. Protein Expression Analyses Immunostaining was performed using a goat polyclonal anti-human UCP1 antibody (sc-6528. along with sample embedding into resin and slicing. LifeSpan BioSciences Inc.). (E and F) 5-FU treatment assay. 50 mM MEK1 inhibitor (PD 98059) (Calbiochem Co. 5 ng/ml VEGFA. 10 ng/ml BMP7. (Tokyo. September 5. Cell Metabolism 16. 20 ng/ml KITLG. 2000). 2012 ª2012 Elsevier Inc.. and 10 mM Akt inhibitor IV (Cat 124011) (Calbiochem Co.Cell Metabolism Brown Adipose Production by Hemopoietin Cocktail Figure 6. Experimental procedure (E) and the results of BM-enucleated cell counts (F) were shown... Maryland.5 ng/ml FLT3LG. and hMSCdWAs (C). Postfixation by 2% osmium tetroxide. hCD45-positive percentages were calculated. was performed by Bio Medical Laboratories Co.5% glutaraldehyde. 394–406. Cat 047-31801) (WAKO Pure Chemical Industries. Santa Cruz Biotechnology. The error bars represent average ± SD (n = 3). Electron Microscopic Examinations Cells were fixed by 2. 2009). hESC/hiPSC-derived spheres were further cultured on gelatin-coated 6-well plates using the above-described serum-free medium supplemented with the hematopoietic cytokine cocktail II (5 ng/ml IGF-II. Western blotting was performed using a rabbit polyclonal UCP1 (Ab10983) (Abcam plc.. Japan) (Saeki et al. 2009). 403 . Hematopoietic Stromal Assays (A) Schematic presentation of the assay.

2012 ª2012 Elsevier Inc. Inc. At day 7. blood samples were taken. and dermal temperature was measured by Thermo GEAR G120/G100 (NEC Avio Infrared Technologies Co. All animal care procedures involved in calorigenic analyses. 30 mmol/kg of isoproterenol (12760. For oral fat tolerance tests.) and anti-human CD33-PE antibody (clone WM53) (BD Biosciences. bone marrow cells were collected from femoral bones and analyzed. Sigma Chemical Co. version 18 (International Business Machines Oxygen Consumption Analyses The adherent culture step of BA differentiation was performed on special 96-well plates (Seahorse Bioscience Inc.or 10-week-old male ICR mice. 30.Cell Metabolism Brown Adipose Production by Hemopoietin Cocktail Figure 7. Ltd. After another 2 hr. Billerica. Typical results of the frontal images under warm and cold conditions were shown. (C and D) Shown are sagittal and axial section images of 18F-FDG-PET/CT under warm (C) and cold conditions (D). After another 4 hr. Floating cells were collected after 7 days. 8..1% gelatin by seeding 30 spheres per well. assessment of lipid and metabolism. Arrows indicate 18F-FDG uptake into vertebrae per se. Ltd.8 ± 5. (B) 18F-FDG-PET/CT. and complied with the procedures of the Guide for the Care and Use of Laboratory Animals of NCGM. Standardized uptake value (SUV) was measured by an expert as described in the Supplemental Experimental Procedures. Ltd.. For myelosuppression recovery assays. Calorigenic Analyses The 1 3 106 of hPSCdBA or immature hPSCs were suspended in 100 ml saline and subcutaneously transplanted into 5-week-old male ICR mice After 24 hr. 394–406. cells were collected from contralateral femoral bone marrow and spleen and subjected to cytometry using an anti-human CD45-FITC (clone J33) (Beckman Coulter Inc. Blood sample were taken after 0. Data are reported as means ± SEM. . and 12 weeks. After 16 hr. Japan). Basel. cord blood CD34+ cells were directly transplanted without coculture. 30 mmol/kg of isoproterenol was administrated from tail vein. Blood glucose concentrations were measured by Accutrend Plus. Hematopoietic Stromal Assays The human cord blood CD34+ cells were cultured on hPSCdBA layers without recombinant cytokines in RPMI1640 medium supplemented with 10% fetal calf serum. After 6. Japan) was orally administrated. 200 ml of olive oil was orally administrated. Assessment of Lipid Metabolism Six-week-old male CR mice were subcutaneously transplanted with 1 3 106 of immature hESC. HoffmannLa Roche. isoproterenol (15 mmol/kg) was administrated. hESCdBA. Wako Pure Chemical Industries. and 2 3 105 cells were transplanted into tibial bone marrow of NOG mice. (E and F) Thoracic vertebra of 3-week-old ICR mice was subjected to HE staining (E) or UCP1 immunostaining (FF). September 5. After another 4 hr. Examinations on BM-BAT (A) Expression of PRDM16 and UCP1 in BM RNA samples of 27-year-old and 41-year-old males and human BM-derived hMSCdWA (hBMMCdWA).8 years of age..) was administrated from the tail vein. and 60 min. which were kept abstained from feed. or hMC-derived WA suspended in 100 ml saline and kept abstained from feed. n = 20) were performed. CA). mice were anesthetized. were measured by Accutrend Plus (F. Japan). For control. 100 mg/kg of 5-FU was intraperitoneally administrated. Tokyo. From day 3 to day 6. After 16 hr. Arrowheads indicate the existence of BA. After another 2 hr. and arrowheads indicate 18 F-FDG uptake into classical paravertebral BA. 18 F-FDG-PET/CT Examinations After careful instruction regarding the study and informed consent to participants. Osaka. ICR mice were subcutaneously transplanted with immature hPSC or hPSCdBA and kept abstained from feed. and hematopoietic stromal assays were approved by the Animal Care and Use Committee of the Research Institute. Oxygen consumption was analyzed by Extracellular Flux Analyzer XF96 (Seahorse Bioscience Inc. Switzerland). 15. Yokohama. isoproterenol (30 mmol/kg) was administrated. and plasma insulin concentrations were measured by mouse insulin ELISA kit (Morinaga Institute of Biological Science. San Jose. After 16 hr. Statistics analyses were performed using SPSS software.. Arrows indicate the 18F-FDG uptake into vertebral BM. PET/CT examinations of healthy young volunteers (24.. Assessment of Glucose Metabolism The 1 3 106 of hESCdBA or hMSCdWA was transplanted to 6. and TG concentrations 404 Cell Metabolism 16. 2 g/kg of glucose (041-00595.) according to the manufacturer’s guidance. The protocol was approved by the institutional review boards of Tenshi College. isoproterenol (30 mmol/kg) was administrated. and blood TG levels were measured every 2 hr. National Center for Global Health and Medicine (NCGM). MA) precoated by 0.

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