BMP7 increases protein synthesis in SW1353 cells and determines rRNA levels in a NKX3-2-dependent manner.

BMP7 is a morphogen capable of counteracting the OA chondrocyte hypertrophic phenotype via NKX3-2. NKX3-2 represses expression of RUNX2, an important transcription factor for chondrocyte hypertrophy. Since RUNX2 has previously been described as an inhibitor for 47S pre-rRNA transcription, we hypothesized that BMP7 positively influences 47S pre-rRNA transcription through NKX3-2, resulting in increased protein translational capacity. Therefor SW1353 cells and human primary chondrocytes were exposed to BMP7 and rRNA (18S, 5.8S, 28S) expression was determined by RT-qPCR. NKX3-2 knockdown was achieved via transfection of a NKX3-2-specific siRNA duplex. Translational capacity was assessed by the SUNsET assay, and 47S pre-rRNA transcription was determined by transfection of a 47S gene promoter-reporter plasmid. BMP7 treatment increased protein translational capacity. This was associated by increased 18S and 5.8S rRNA and NKX3-2 mRNA expression, as well as increased 47S gene promotor activity. Knockdown of NKX3-2 led to increased expression of RUNX2, accompanied by decreased 47S gene promotor activity and rRNA expression, an effect BMP7 was unable to restore. Our data demonstrate that BMP7 positively influences protein translation capacity of SW1353 cells and chondrocytes. This is likely caused by an NKX3-2-dependent activation of 47S gene promotor activity. This finding connects morphogen-mediated changes in cellular differentiation to an aspect of ribosome biogenesis via key transcription factors central to determining the chondrocyte phenotype.

Overexpression of bone morphogenetic protein 7 reduces oligodendrocytes loss and promotes functional recovery after spinal cord injury.

Spinal cord injury (SCI), as a severe disease with no effective therapeutic measures, has always been a hot topic for scientists. Bone morphogenetic protein 7 (BMP7), as a multifunctional cytokine, has been reported to exert protective effects on the nervous system. The present study aimed to investigate the neuroprotective effect and the potential mechanisms of BMP7 on rats that suffered SCI. Rat models of SCI were established by the modified Allen' s method. Adeno-associated virus (AAV) was injected at T9 immediately before SCI to overexpress BMP7. Results showed that the expression of BMP7 decreased in the injured spinal cords that were at the same time demyelinated. AAV-BMP7 partly reversed oligodendrocyte (OL) loss, and it was beneficial to maintain the normal structure of myelin. The intervention group showed an increase in the number of axons and Basso-Beattie-Bresnahan scores. Moreover, double-labelled immunofluorescence images indicated p-Smad1/5/9 and p-STAT3 in OLs induced by BMP7 might be involved in the protective effects of BMP7. These findings suggest that BMP7 may be a feasible therapy for SCI to reduce demyelination and promote functional recovery.

Bone morphogenetic protein-7 inhibits endothelial-to-mesenchymal transition in primary human umbilical vein endothelial cells and mouse model of systemic sclerosis via Akt/mTOR/p70S6K pathway.

BACKGROUND: Systemic sclerosis (SSc) is an autoimmune inflammatory and vascular disorder that causes tissue fibrosis of the skin and internal organs. Endothelial-to-mesenchymal transition (EndoMT) has been considered an important mechanism in the pathogenesis of vascular remodeling in SSc. Recent studies suggested that bone morphogenic protein 7 (BMP-7) has anti-fibrotic effects in several fibrotic diseases. OBJECTIVES: To investigate the mechanism of BMP-7 in inhibiting TGF-ß-induced EndoMT in systemic sclerosis (SSc). METHODS: Skin tissues of both healthy controls and SSc patients were detected the distribution of BMP-7. TGF-ß was applied to induce the EndoMT model of human umbilical vein endothelial cells (HUVECs), and bleomycin was used to established the SSc mouse model. After treatment of BMP-7, the protein levels of endothelial specific markers, mesenchymal cell products, transcription factors and Akt signal pathway were examined by western blotting, immunofluorescence or immunohistochemistry both in vivo and in vitro. RESULTS: The expression of BMP-7 was decreased in the basal layer of epidermis and dermis of SSc patients. EndoMT in TGF-ß-treated HUVECs and skins of SSc mouse model were markedly attenuated after treatment with rh-BMP-7. Moreover, Akt/mTOR/p70S6K phosphorylation was involved in EndoMT and BMP-7 suppressed TGF-ß- or bleomycin-induced theses phosphorylation in HUVECs or SSc mouse model. CONCLUSION: BMP-7 reduced the production of TGF-ß-induced EndoMT in HUVECs and SSc mouse model through Akt/mTOR/p70S6K signaling pathway. These findings suggested that BMP-7 could be employed as a promising antifibrotic therapy for SSc.

Bone Morphogenetic Protein-7 (BMP-7) Promotes Neuronal Differentiation of Bone Marrow Mesenchymal Stem Cells (BMSCs) In Vitro.

This study is aimed at investigating the effects of bone morphogenetic protein-7 (BMP-7) on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into neuron-like cells in vitro. The rat BMSCs were isolated and identified, which were divided into the control, empty, recombinant rhBMP-7 transfection, and Lv-BMP-7 transfection groups. BMSCs were induced under different conditions. CCK-8 assay was performed to detect cell proliferation. ALP was used to detect cell activity. Cellular morphology after induction was observed. Immunofluorescence was conducted to detect the expression and location of nerve cell markers. Quantitative real-time PCR and Western blot analysis were performed to detect the mRNA and protein expression levels, respectively. The rhBMP-7 and Lv-BMP-7 promoted the proliferation of BMSCs, accompanied with increased ALP activities. Morphological observations revealed that rhBMP-7 and Lv-BMP-7 induced BMSCs to differentiate into neuron-like cells. Immunofluorescence revealed that the rhBMP-7 and Lv-BMP-7 groups showed positive expression of MAP-2 and Nfh in BMSCs. MAP-2 was mainly distributed in the cell body and cellular protrusion, while Nfh was mainly distributed in the cytoplasm and cell protrusion. Positive mRNA and protein expressions of MAP-2 and Nfh were observed in the cells of the rhBMP-7 and Lv-BMP-7 groups, and the expression levels were significantly higher than the control and empty groups. Both exogenous BMP-7 (rhBMP-7) and endogenous BMP-7 (Lv-BMP-7) can induce BMSCs to differentiate into neuron-like cells highly expressing the neuronal markers MAP-2 and Nfh.

Effective reconstruction of functional organotypic kidney spheroid for in vitro nephrotoxicity studies.

Stable and reproducible kidney cellular models could accelerate our understanding of diseases, help therapeutics development, and improve nephrotoxicity screenings. Generation of a reproducible in vitro kidney models has been challenging owing to the cellular heterogeneity and structural complexity of the kidney. We generated mixed immortalized cell lines that stably maintained their characteristic expression of renal epithelial progenitor markers for the different lineages of kidney cellular compartments via the BMP7 signaling pathway from a mouse and a human whole kidney. These cells were used to generate functional and matured kidney spheroids containing multiple renal lineages, such as the proximal tubule, loop of Henle, distal tubules, and podocytes, using extracellular matrix and physiological force, named spheroid-forming unit (SFU). They expressed all apical and basolateral transporters that are important for drug metabolism and displayed key functional aspects of the proximal tubule, including protein endocytosis and increased gamma-glutamyltransferase activity, and cyclic AMP responded to external cues, such as parathyroid hormone. Following exposure, cells fluxed and took up drugs via proximal tubule-specific apical or basolateral transporters, and displayed increased cell death and expression of renal injury marker. Here, we developed a new differentiation method to generate kidney spheroids that structurally recapitulate important features of the kidney effectively and reproducibly using mixed immortalized renal cells, and showed their application for renal toxicity studies.

Nuclear hyaluronidase 2 drives alternative splicing of CD44 pre-mRNA to determine profibrotic or antifibrotic cell phenotype.

The cell surface protein CD44 is involved in diverse physiological processes, and its aberrant function is linked to various pathologies such as cancer, immune dysregulation, and fibrosis. The diversity of CD44 biological activity is partly conferred by the generation of distinct CD44 isoforms through alternative splicing. We identified an unexpected function for the ubiquitous hyaluronan-degrading enzyme, hyaluronidase 2 (HYAL2), as a regulator of CD44 splicing. Standard CD44 is associated with fibrotic disease, and its production is promoted through serine-arginine-rich (SR) protein-mediated exon exclusion. HYAL2 nuclear translocation was stimulated by bone morphogenetic protein 7, which inhibits the myofibroblast phenotype. Nuclear HYAL2 displaced SR proteins from the spliceosome, thus enabling HYAL2, spliceosome components (U1 and U2 small nuclear ribonucleoproteins), and CD44 pre-mRNA to form a complex. This prevented double-exon splicing and facilitated the inclusion of CD44 exons 11 and 12, which promoted the accumulation of the antifibrotic CD44 isoform CD44v7/8 at the cell surface. These data demonstrate previously undescribed mechanisms regulating CD44 alternative splicing events that are relevant to the regulation of cellular phenotypes in progressive fibrosis.

Branching morphogenesis in the developing kidney is governed by rules that pattern the ureteric tree.

Metanephric kidney development is orchestrated by the iterative branching morphogenesis of the ureteric bud. We describe an underlying patterning associated with the ramification of this structure and show that this pattern is conserved between developing kidneys, in different parts of the organ and across developmental time. This regularity is associated with a highly reproducible branching asymmetry that is consistent with locally operative growth mechanisms. We then develop a class of tip state models to represent elaboration of the ureteric tree and describe rules for 'half-delay' branching morphogenesis that describe almost perfectly the patterning of this structure. Spatial analysis suggests that the observed asymmetry may arise from mutual suppression of bifurcation, but not extension, between the growing ureteric tips, and demonstrates that disruption of patterning occurs in mouse mutants in which the distribution of tips on the surface of the kidney is altered. These findings demonstrate that kidney development occurs by way of a highly conserved reiterative pattern of asymmetric bifurcation that is governed by intrinsic and locally operative mechanisms.

Cbfß2 deficiency preserves Langerhans cell precursors by lack of selective TGFß receptor signaling.

The mouse Langerhans cell (LC) network is established through the differentiation of embryonic LC precursors. BMP7 and TGFß1 initiate cellular signaling that is essential for inducing LC differentiation and preserving LCs in a quiescent state, respectively. Here we show that loss of Cbfß2, one of two RNA splice variants of the Cbfb gene, results in long-term persistence of embryonic LC precursors after their developmental arrest at the transition into the EpCAM+ stage. This phenotype is caused by selective loss of BMP7-mediated signaling essential for LC differentiation, whereas TGFßR signaling is intact, maintaining cells in a quiescent state. Transgenic Cbfß2 expression at the neonatal stage, but not at the adult stage, restored differentiation from Cbfß2-deficient LC precursors. Loss of developmental potential in skin-residential precursor cells was accompanied by diminished BMP7-BMPR1A signaling. Collectively, our results reveal an essential requirement for the Cbfß2 variant in LC differentiation and provide novel insight into how the establishment and homeostasis of the LC network is regulated.

Bone Morphogenetic Protein-7 Suppresses TGFß2-Induced Epithelial-Mesenchymal Transition in the Lens: Implications for Cataract Prevention.

Purpose: Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is a key pathologic mechanism underlying cataract. Two members of the transforming growth factor-ß (TGFß) superfamily, TGFß and bone morphogenetic protein-7 (BMP-7) have functionally distinct roles in EMT. While TGFß is a potent inducer of EMT, BMP-7 counteracts the fibrogenic activity of TGFß. We examine the modulating effect of BMP-7 on TGFß-induced EMT in LECs. Methods: Rat lens epithelial explants were treated exogenously with TGFß2 alone or in combination with BMP-7 for up to 5 days. Expression levels of E-cadherin, ß-catenin, α-smooth muscle actin (α-SMA), and phosphorylated downstream Smads were determined using immunofluorescence and Western blotting. Reverse transcriptase quantitative PCR (RT-qPCR) was used to study gene expression levels of EMT markers and downstream BMP target genes, including the Inhibitors of differentiation (Id). Results: Transforming growth factor-ß2 induced LECs to transdifferentiate into myofibroblastic cells. Addition of BMP-7 suppressed TGFß2-induced α-SMA protein levels and mesenchymal gene expression, with retention of E-cadherin and ß-catenin expression to the cell membrane. Addition of BMP-7 prevented lens capsular wrinkling and cellular loss associated with TGFß2-induced EMT over the 5-day treatment period. The inhibitory effect of BMP-7 was accompanied by an early induction of pSmad1/5 and suppression of TGFß2-induced pSmad2/3. Treatment with TGFß2 alone suppressed gene expression of Id2/3 and addition of BMP-7 restored Id2/3 expression. Conclusions: Exogenous administration of BMP-7 abrogated TGFß2-induced EMT in rat lens epithelial explants. Understanding the complex interplay between the TGFß- and BMP-7-associated Smad signaling pathways and their downstream target genes holds therapeutic promise in cataract prevention.

Bone morphogenetic protein-2 and tumor growth: Diverse effects and possibilities for therapy.

Concern regarding safety with respect to the clinical use of human bone morphogenetic protein-2 (BMP-2) has become an increasingly controversial topic. The role of BMP-2 in carcinogenesis is of particular concern. Although there have been many studies of this topic, the results have been contradictory and confusing. We conducted a systematic review of articles that are relevant to the relationship or effect of BMP-2 on all types of tumors and a total of 97 articles were included. Studies reported in these articles were classified into three major types: "expression studies", "in vitro studies", and "in vivo studies". An obvious pattern was that those works that hypothesize an inhibitory effect for BMP-2 most often examined only the proliferative properties of the tumor cells. This subset of studies also contained an extraordinary number of contradictory findings which made drawing a reliable general conclusion impossible. In general, we support a pro-tumorigenesis role for BMP-2 based on the data from these in vitro cell studies and in vivo animal studies, however, more clinical studies should be carried out to help make a firm conclusion.

Suppression of hepatic stellate cell activation through downregulation of gremlin1 expression by the miR-23b/27b cluster.

The imbalance between transforming growth factor ß and bone morphogenetic protein 7 signaling pathways is a critical step in promoting hepatic stellate cell activation during hepatic fibrogenesis. Gremlin1 may impair the balance. Something remains unclear about the regulatory mechanisms of gremlin1 action on hepatic stellate cell activation and hepatic fibrosis. In the current study, gremlin1 overexpression promotes activation of hepatic stellate cells. Knockdown of gremlin1 with siRNAs suppresses hepatic stellate cell activation and attenuates hepatic fibrosis in rat model. Our results also show that miR-23b/27b cluster members bind to 3'-untranslated region of gremlin1 resulting in reduction of transforming growth factor ß, α-smooth muscle actin and collagenI α1/2 gene expression. Our findings suggest that gremlin1 promotes hepatic stellate cell activation and hepatic fibrogenesis through impairment of the balance between transforming growth factor ß and bone morphogenetic protein 7 signaling pathways. The miR-23b/27b cluster suppresses activation of hepatic stellate cells through binding gremlin1 to rectify the imbalance.

Effect on Clinical Outcome and Growth Factor Synthesis With Adjunctive Use of Pulsed Electromagnetic Fields for Fifth Metatarsal Nonunion Fracture: A Double-Blind Randomized Study.

BACKGROUND: Electromagnetic bone growth stimulators have been found to biologically enhance the bone healing environment, with upregulation of numerous growth factors. The purpose of the study was to quantify the effect, in vivo, of pulsed electromagnetic fields (PEMFs) on growth factor expression and healing time in fifth metatarsal nonunions. METHODS: This was a prospective, randomized, double-blind trial of patients, cared for by 2 fellowship-trained orthopedic foot and ankle surgeons. Inclusion criteria consisted of patients between 18 and 75 years old who had been diagnosed with a fifth metatarsal delayed or nonunion, with no progressive signs of healing for a minimum of 3 months. Eight patients met inclusion criteria and were randomized to receive either an active stimulation or placebo PEMF device. Each patient then underwent an open biopsy of the fracture site and was fitted with the appropriate PEMF device. The biopsy was analyzed for messenger-ribonucleic acid (mRNA) levels using quantitative competitive reverse transcription polymerase chain reaction (QT-RT-PCR). Three weeks later, the patient underwent repeat biopsy and open reduction and internal fixation of the nonunion site. The patients were followed at 2- to 4-week intervals with serial radiographs and were graded by the number of cortices of healing. RESULTS: All fractures healed, with an average time to complete radiographic union of 14.7 weeks and 8.9 weeks for the inactive and active PEMF groups, respectively. A significant increase in placental growth factor (PIGF) level was found after active PEMF treatment (P = .043). Other factors trended higher following active PEMF including brain-derived neurotrophic factor (BDNF), bone morphogenetic protein (BMP) -7, and BMP-5. CONCLUSION: The adjunctive use of PEMF for fifth metatarsal fracture nonunions produced a significant increase in local placental growth factor. PEMF also produced trends toward higher levels of multiple other factors and faster average time to radiographic union compared to unstimulated controls. LEVEL OF EVIDENCE: Level I, prospective randomized trial.

BMP-7 Attenuates TGF-ß1-Induced Fibronectin Secretion and Apoptosis of NRK-52E Cells by the Suppression of miRNA-21.

Bone morphogenetic protein-7 (BMP-7) inhibited the pathogenesis of renal injury in response to a variety of stimuli. However, little is known about the molecular regulation and mechanism of endogenous BMP-7 and its renoprotective functions. This study examined the regulation of BMP-7 and its role in the fibronectin secretion and apoptosis of NRK-52E cells resulting from transforming growth factor-ß1 (TGF-ß1) in vitro. Results showed that TGF-ß1 promoted factor-associated suicide (FAS), FAS ligand (FASL), fibronectin (FN), and miRNA-21 expression, while it downregulated phospho-Smad1 (pSmad1), pSmad5, and pSmad8 expressions in NRK-52E cells. In contrast, BMP-7 alleviated TGF-ß1-induced cell apoptosis, inhibited TGF-ß1-induced higher expression of miRNA-21 and FN, and enhanced TGF-ß1-attenuated phosphorylation of Smad1, Smad5, and Smad8. Furthermore, a chemical inhibitor of miRNA-21 also negatively affected TGF-ß1-induced apoptosis and FN secretion. On the other hand, overexpression of miRNA-21 counteracted the inhibitory effect of BMP-7 on TGF-ß1-induced FN secretion and apoptosis. However, BMP-7 showed no effects on TGF-ß1-induced FN secretion and apoptosis following knockdown of miRNA-21. Taken together, these findings demonstrated that BMP-7 might inhibit TGF-ß1-induced FN secretion and apoptosis by the suppression of miRNA-21 in NRK-52E cells.

Roles of the kidney in the formation, remodeling and repair of bone.

The relationship between the kidney and bone is highly complex, and the kidney plays an important role in the regulation of bone development and metabolism. The kidney is the major organ involved in the regulation of calcium and phosphate homeostasis, which is essential for bone mineralization and development. Many substances synthesized by the kidney, such as 1,25(OH)2D3, Klotho, bone morphogenetic protein-7, and erythropoietin, are involved in different stages of bone formation, remodeling and repair. In addition, some cytokines which can be affected by the kidney, such as osteoprotegerin, sclerostin, fibroblast growth factor -23 and parathyroid hormone, also play important roles in bone metabolism. In this paper, we summarize the possible effects of these kidney-related cytokines on bone and their possible mechanisms. Most of these cytokines can interact with one another, constituting an intricate network between the kidney and bone. Therefore, kidney diseases should be considered among patients presenting with osteodystrophy and disturbances in bone and mineral metabolism, and treatment for renal dysfunction may accelerate their recovery.

BMP signalling in skeletal development, disease and repair.

Since the identification in 1988 of bone morphogenetic protein 2 (BMP2) as a potent inducer of bone and cartilage formation, BMP superfamily signalling has become one of the most heavily investigated topics in vertebrate skeletal biology. Whereas a large part of this research has focused on the roles of BMP2, BMP4 and BMP7 in the formation and repair of endochondral bone, a large number of BMP superfamily molecules have now been implicated in almost all aspects of bone, cartilage and joint biology. As modulating BMP signalling is currently a major therapeutic target, our rapidly expanding knowledge of how BMP superfamily signalling affects most tissue types of the skeletal system creates enormous potential to translate basic research findings into successful clinical therapies that improve bone mass or quality, ameliorate diseases of skeletal overgrowth, and repair damage to bone and joints. This Review examines the genetic evidence implicating BMP superfamily signalling in vertebrate bone and joint development, discusses a selection of human skeletal disorders associated with altered BMP signalling and summarizes the status of modulating the BMP pathway as a therapeutic target for skeletal trauma and disease.

BMPs are direct triggers of interdigital programmed cell death.

During vertebrate embryogenesis the interdigital mesenchyme is removed by programmed cell death (PCD), except in species with webbed limbs. Although bone morphogenetic proteins (BMPs) have long been known to be players in this process, it is unclear if they play a direct role in the interdigital mesenchyme or if they only act indirectly, by affecting fibroblast growth factor (FGF) signaling. A series of genetic studies have shown that BMPs act indirectly by regulating the withdrawal of FGF activity from the apical ectodermal ridge (AER); this FGF activity acts as a cell survival factor for the underlying mesenchyme. Other studies using exogenous factors to inhibit BMP activity in explanted mouse limbs suggest that BMPs do not act directly in the mesenchyme. To address the question of whether BMPs act directly, we used an interdigit-specific Cre line to inactivate several genes that encode components of the BMP signaling pathway, without perturbing the normal downregulation of AER-FGF activity. Of three Bmps expressed in the interdigital mesenchyme, Bmp7 is necessary for PCD, but Bmp2 and Bmp4 both have redundant roles, with Bmp2 being the more prominent player. Removing BMP signals to the interdigit by deleting the receptor gene, Bmpr1a, causes a loss of PCD and syndactyly, thereby unequivocally proving that BMPs are direct triggers of PCD in this tissue. We present a model in which two events must occur for normal interdigital PCD: the presence of a BMP death trigger and the absence of an FGF survival activity. We demonstrate that neither event is required for formation of the interdigital vasculature, which is necessary for PCD. However, both events converge on the production of reactive oxygen species that activate PCD.

Febuxostat Prevents Renal Interstitial Fibrosis by the Activation of BMP-7 Signaling and Inhibition of USAG-1 Expression in Rats.

BACKGROUND: Renal interstitial fibrosis (RIF) is a common pathology associated with end-stage renal diseases. The activation of bone morphogenetic protein-7 (BMP-7)-Smad1/5/8 pathway seems to alleviate RIF. Uterine sensitization-associated gene-1 (USAG-1), a kidney-specific BMPs antagonist, is associated with the development and prognosis of several renal diseases. Febuxostat is a xanthine oxidase inhibitor that can attenuate the renal dysfunction of patients. The purpose of this study was to investigate the effects of febuxostat on renal fibrosis and to clarify the mechanisms underlying these effects. METHODS: Rats were randomly divided into 6 groups termed a sham-operated group, a unilateral ureteral obstruction (UUO) group, 3 doses of febuxostat groups (low, intermediate and high doses) and a sham group treated with high-dose febuxostat. After 14 days, renal function, relative kidney weight, accumulation of glycogen and collagens were examined by different methods. Expression of α-SMA, transforming growth factor-ß1 (TGF-ß1), BMP-7 and USAG-1 was detected by western blotting and RT-PCR, respectively. The phosphorylation level of Smad1/5/8 was also quantified by western blotting. RESULTS: The renal function was declined, and large amounts of glycogen and collagens were deposited in the kidneys of UUO rats compared with the rats in the sham group. Besides, expression of α-SMA and USAG-1 in these kidneys was elevated, and the TGF-ß1 was also activated, while the BMP-7-Smad1/5/8 pathway was inhibited. Febuxostat reversed the changes stated earlier, exhibiting protective effects on RIF induced by UUO. CONCLUSION: Febuxostat was able to attenuate RIF caused by UUO, which was associated with the activation of BMP-7-Smad1/5/8 pathway and the inhibition of USAG-1 expression in the kidneys of UUO rats.

A Combination of Culture Conditions and Gene Expression Analysis Can Be Used to Investigate and Predict hES Cell Differentiation Potential towards Male Gonadal Cells.

Human embryonic stem cell differentiation towards various cell types belonging to ecto-, endo- and mesodermal cell lineages has been demonstrated, with high efficiency rates using standardized differentiation protocols. However, germ cell differentiation from human embryonic stem cells has been very inefficient so far. Even though the influence of various growth factors has been evaluated, the gene expression of different cell lines in relation to their differentiation potential has not yet been extensively examined. In this study, the potential of three male human embryonic stem cell lines to differentiate towards male gonadal cells was explored by analysing their gene expression profiles. The human embryonic stem cell lines were cultured for 14 days as monolayers on supporting human foreskin fibroblasts or as spheres in suspension, and were differentiated using BMP7, or spontaneous differentiation by omitting exogenous FGF2. TLDA analysis revealed that in the undifferentiated state, these cell lines have diverse mRNA profiles and exhibit significantly different potentials for differentiation towards the cell types present in the male gonads. This potential was associated with important factors directing the fate of the male primordial germ cells in vivo to form gonocytes, such as SOX17 or genes involved in the NODAL/ACTIVIN pathway, for example. Stimulation with BMP7 in suspension culture resulted in up-regulation of cytoplasmic SOX9 protein expression in all three lines. The observation that human embryonic stem cells differentiate towards germ and somatic cells after spontaneous and BMP7-induced stimulation in suspension emphasizes the important role of somatic cells in germ cell differentiation in vitro.

Transcriptional Regulatory Circuits Controlling Brown Fat Development and Activation.

Brown and beige adipose tissue is specialized for heat production and can be activated to reduce obesity and metabolic dysfunction in animals. Recent studies also have indicated that human brown fat activity levels correlate with leanness. This has revitalized interest in brown fat biology and has driven the discovery of many new regulators of brown fat development and function. This review summarizes recent advances in our understanding of the transcriptional mechanisms that control brown and beige fat cell development.