Adipocyte Lineage Research Articles

QS1: Creeping Fat Adipocytes Drive Intestinal Fibrosis Through Adipocyte-to-fibroblast Conversion in a Novel Model of Inflammatory Bowel Disease

Purpose:Crohn’s disease (CD) is a subtype of inflammatory bowel disease (IBD) characterized by patchy, transmural inflammation throughout the digestive tract and creeping fat formation. Thirty percent of CD patients develop strictures, eighty percent of which will require surgery. Creeping fat is associated with stricture formation, but its role in intestinal fibrosis remains unclear. Here, we present a novel surgical model of intestinal fibrosis and show by lineage tracing that creeping fat adipocytes convert to fibroblasts that contribute to fibrosis.Methods:We developed a novel surgical model of IBD that avoids the chronic use of caustic agents by creating a longitudinal, anti-mesenteric colotomy in the mouse transverse colon that is closed transversally. We performed Masson’s trichrome staining to assess collagen deposition. Finally, we performed lineage tracing of mature adipocytes in Adiponectin-Cre; mTmG mice and characterized adipocyte-derived cells by immunostaining.Results:Our surgical model mimics key features of human strictures, including the formation of creeping fat around the injury site, increased bowel wall thickness, collagen deposition, and transmural adipocyte infiltration by post-operative day (POD) 7. Immunostaining for adipocyte and fibroblast markers confirmed the presence of transmural adipocytes adjacent to fibroblasts. Lineage tracing of mature adipocytes in Adiponectin-Cre; mTmG mice revealed adipocyte-derived cells that infiltrate the wound site that lose expression of adipocyte lineage markers, gain expression of fibroblast markers, produce collagen, and respond to TGF-β signaling.Conclusions:Our novel surgical colotomy model represents a viable approach to study intestinal fibrosis and creeping fat without the long-term use of caustic agents. Lineage tracing of mature creeping fat adipocytes demonstrates that adipocytes convert to fibroblasts that infiltrate the injury site and participate in fibrotic responses. Taken together, these findings suggest that creeping fat contributes to intestinal fibrosis in part through the local conversion of adipocytes to fibroblasts.

A-769662 stimulates the differentiation of bone marrow-derived mesenchymal stem cells into osteoblasts via AMP-activated protein kinase-dependent mechanism.

AMP-activated protein kinase (AMPK) signaling shows an important role in energy metabolism and has recently been involved in osteogenic and adipogenic differentiation. In this study we aimed to investigate the role of AMPK activator, A-769662, in regulating the differentiation of mesenchymal stem cells derived from bone marrow (BMSCs) into osteoblastic and adipocytic cell lineage. The effect of A-769662 on osteogenesis was assessed by quantitative alkaline phosphatase (ALP) activity, matrix mineralization stained with Alizarin red, and gene expression analysis by quantitative polymerase chain reaction (qPCR). Adipogenesis was determined by Oil Red O staining for fat droplets and qPCR analysis of adipogenic markers. A-769662 activated the phosphorylation of AMPKα1 during the osteogenesis of mBMSCs as revealed by western blot analysis. A-769662 promoted the early stage of the commitment of mouse (m) BMSCs differentiation into osteoblasts, while inhibiting their differentiation into adipocytes in a dose-dependent manner. The effects of A-769662 on stimulating osteogenesis and inhibiting adipogenesis of mBMSCs were significantly eliminated in the presence of either AMPKα1 siRNA or Compound C, an inhibitor of AMPK pathway. In conclusion, we identified A-769662 as a new compound that promotes the commitment of BMSCs into osteoblasts versus adipocytes via AMPK-dependent mechanism. Thus our data show A-769662 as a potential osteo-anabolic drug for treatment of osteoporosis.

Abstract 2974: Loss of TSC1 or TSC2 drives lineage infidelity and hamartoma formation in a renal organoid model of angiomyolipoma

Abstract Renal angiomyolipomas (R-AMLs) are hamartomatous kidney tumors which stain positively for markers of adipocytic, vascular, smooth muscle, and melanocytic lineages. These lesions possess loss of function mutations in either TSC1 or TSC2, which are canonical negative regulators of mTORC1 signaling. To date, there exists no in vitro or in vivo model which faithfully recapitulates the architectural and molecular complexity of R-AMLs. Considering these lesions can be detected very early in life- even congenitally- we hypothesized they arise as a consequence of aberrant tissue development. To test this hypothesis, we generated TSC1-/- and TSC2-/- mutants in four human pluripotent stem cell (hPSC) backgrounds using CRISPR/Cas9 genome engineering. Wild type hPSCs differentiated into renal organoids express markers of the glomerulus, proximal and distal tubules, in a topology that resembles human nephron patterning. Remarkably, wild type renal organoids downregulate mTORC1 signaling compared to adjacent undifferentiated cells. In contrast, both TSC1-/- and TSC2-/- hPSCs exhibit substantial lineage infidelity upon differentiation, staining positively for adipocytic and melanocytic markers which are absent in matched wild type controls. Additionally, knockout lines formed nodular growths with disorganized architecture, resembling the hamartomatous organization of R-AML lesions. These lesions exhibit hyperactive mTORC1 signaling, consistent with human pathology. Together, these data suggest three primary findings: loss of TSC1/2 drives lineage infidelity; TSC1/2 may be required for architectural organization of the kidney parenchyma; and a developmental approach to R-AML modelling may best recapitulate the human disease. Citation Format: Adam Pietrobon, Sean P. Delaney, Carole Doré, Julien Yockell-Lelievre, Lisa Julian, William L. Stanford. Loss of TSC1 or TSC2 drives lineage infidelity and hamartoma formation in a renal organoid model of angiomyolipoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2974.

Identification of thermogenic adipocyte lineages.

Identification of thermogenic adipocyte lineages.

Runx1 is a central regulator of osteogenesis for bone homeostasis by orchestrating BMP and WNT signaling pathways.

Runx1 is highly expressed in osteoblasts, however, its function in osteogenesis is unclear. We generated mesenchymal progenitor-specific (Runx1f/fTwist2-Cre) and osteoblast-specific (Runx1f/fCol1α1-Cre) conditional knockout (Runx1 CKO) mice. The mutant CKO mice with normal skeletal development displayed a severe osteoporosis phenotype at postnatal and adult stages. Runx1 CKO resulted in decreased osteogenesis and increased adipogenesis. RNA-sequencing analysis, Western blot, and qPCR validation of Runx1 CKO samples showed that Runx1 regulates BMP signaling pathway and Wnt/β-catenin signaling pathway. ChIP assay revealed direct binding of Runx1 to the promoter regions of Bmp7, Alk3, and Atf4, and promoter mapping demonstrated that Runx1 upregulates their promoter activity through the binding regions. Bmp7 overexpression rescued Alk3, Runx2, and Atf4 expression in Runx1-deficient BMSCs. Runx2 expression was decreased while Runx1 was not changed in Alk3 deficient osteoblasts. Atf4 overexpression in Runx1-deficient BMSCs did not rescue expression of Runx1, Bmp7, and Alk3. Smad1/5/8 activity was vitally reduced in Runx1 CKO cells, indicating Runx1 positively regulates the Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 signaling pathway. Notably, Runx1 overexpression in Runx2-/- osteoblasts rescued expression of Atf4, OCN, and ALP to compensate Runx2 function. Runx1 CKO mice at various osteoblast differentiation stages reduced Wnt signaling and caused high expression of C/ebpα and Pparγ and largely increased adipogenesis. Co-culture of Runx1-deficient and wild-type cells demonstrated that Runx1 regulates osteoblast−adipocyte lineage commitment both cell-autonomously and non-autonomously. Notably, Runx1 overexpression rescued bone loss in OVX-induced osteoporosis. This study focused on the role of Runx1 in different cell populations with regards to BMP and Wnt signaling pathways and in the interacting network underlying bone homeostasis as well as adipogenesis, and has provided new insight and advancement of knowledge in skeletal development. Collectively, Runx1 maintains adult bone homeostasis from bone loss though up-regulating Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 and WNT/β-Catenin signaling pathways, and targeting Runx1 potentially leads to novel therapeutics for osteoporosis.

Effect of Low-Intensity Vibration on Bone Strength, Microstructure, and Adiposity in Pre-Osteoporotic Postmenopausal Women: A Randomized Placebo-Controlled Trial

ABSTRACT There has been evidence that cyclical mechanical stimulation may be osteogenic, thus providing opportunities for nonpharmacological treatment of degenerative bone disease. Here, we applied this technology to a cohort of postmenopausal women with varying bone mineral density (BMD) T-scores at the total hip (−0.524 ± 0.843) and spine (−0.795 ± 1.03) to examine the response to intervention after 1 year of daily treatment with 10 minutes of vibration therapy in a randomized double-blinded trial. The device operates either in an active mode (30 Hz and 0.3 g) or placebo. Primary endpoints were changes in bone stiffness at the distal tibia and marrow adiposity of the vertebrae, based on 3 Tesla high-resolution MRI and spectroscopic imaging, respectively. Secondary outcome variables included distal tibial trabecular microstructural parameters and vertebral deformity determined by MRI, volumetric and areal bone densities derived using peripheral quantitative computed tomography (pQCT) of the tibia, and dual-energy X-ray absorptiometry (DXA)-based BMD of the hip and spine. Device adherence was 83% in the active group (n = 42) and 86% in the placebo group (n = 38) and did not differ between groups (p = .7). The mean 12-month changes in tibial stiffness in the treatment group and placebo group were +1.31 ± 6.05% and −2.55 ± 3.90%, respectively (group difference 3.86%, p = .0096). In the active group, marrow fat fraction significantly decreased after 12 months of intervention (p = .0003), whereas no significant change was observed in the placebo group (p = .7; group difference −1.59%, p = .029). Mean differences of the changes in trabecular bone volume fraction (p = .048) and erosion index (p = .044) were also significant, as was pQCT-derived trabecular volumetric BMD (vBMD; p = .016) at the tibia. The data are commensurate with the hypothesis that vibration therapy is protective against loss in mechanical strength and, further, that the intervention minimizes the shift from the osteoblastic to the adipocytic lineage of mesenchymal stem cells. © 2020 American Society for Bone and Mineral Research (ASBMR).

Low-dose Bisphenol-A Promotes Epigenetic Changes at Pparγ Promoter in Adipose Precursor Cells.

Exposure to endocrine-disrupting chemicals such as Bisphenol-A (BPA) is associated with an increase in obesity prevalence. Diet is the primary cause of human exposure to this contaminant. BPA promotes obesity by inducing adipocyte dysfunction and altering adipogenesis. Contradictory evidence and unanswered questions are reported in the literature concerning the BPA effects on adipogenesis. To clarify this issue, we tested the effects of prolonged low-dose BPA exposure on different phases of adipogenesis in committed 3T3L1 and uncommitted NIH3T3 preadipocytes. Our findings show that BPA effects on the adipogenesis are mediated by epigenetic mechanisms by reducing peroxisome proliferator-activated receptor gamma (Pparγ) promoter methylation in preadipocytes. Nevertheless, in BPA-exposed 3T3L1, Pparγ expression only transiently increases as lipid accumulation at day 4 of differentiation, without altering the adipogenic potential of the precursor cells. In the absence of differentiation mix, BPA does not make the 3T3L1 an in vitro model of spontaneous adipogenesis and the effects on the Pparγ expression are still limited at day 4 of differentiation. Furthermore, BPA exposure does not commit the NIH3T3 to the adipocyte lineage, although Pparγ overexpression is more evident both in preadipocytes and during the adipocyte differentiation. Interestingly, termination of the BPA exposure restores the Pparγ promoter methylation and inflammatory profile of the 3T3L1 cells. This study shows that BPA induces epigenetic changes in a key adipogenic gene. These modifications are reversible and do not affect preadipocyte commitment and/or differentiation. We identify an alternative transcriptional mechanism by which BPA affects gene expression and demonstrate how the challenge of preventing exposure is fundamental for human health.

Recapitulation of Neural Crest Specification and EMT via Induction from Neural Plate Border-like Cells

SummaryNeural crest cells (NCCs) contribute to several tissues during embryonic development. NCC formation depends on activation of tightly regulated molecular programs at the neural plate border (NPB) region, which initiate NCC specification and epithelial-to-mesenchymal transition (EMT). Although several approaches to investigate NCCs have been devised, these early events of NCC formation remain largely unknown in humans, and currently available cellular models have not investigated EMT. Here, we report that the E6 neural induction protocol converts human induced pluripotent stem cells into NPB-like cells (NBCs), from which NCCs can be efficiently derived. NBC-to-NCC induction recapitulates gene expression dynamics associated with NCC specification and EMT, including downregulation of NPB factors and upregulation of NCC specifiers, coupled with other EMT-associated cell-state changes, such as cadherin modulation and activation of TWIST1 and other EMT inducers. This strategy will be useful in future basic or translational research focusing on these early steps of NCC formation.

Abstract 2476: Role of retinoblastoma gene in maintenance of osteoblast function and communication

Abstract Retinoblastoma gene (Rb1) loss of function mutations is commonly seen in osteosarcomas resulting in poorly differentiated tumors. In addition to cell cycle regulation, Rb1 function has been shown to be important in cell fate determination. It is known that loss of Rb1 function in mesenchymal stem cells inhibits osteoblast differentiation and predisposes them towards adipocytic lineage. Recent studies have suggested a role for Rb1 in cell adhesion but mechanistic details are missing. As cadherin 11 (Cad11) and gap junctional protein connexin43 (Cx43), play important roles in adhesion and communication in osteoblasts, we studied these proteins in MC3T3-E1 mouse calvaria osteoblast cell line. We compared osteoblasts with reduced Rb1 expression to vector-transfected osteoblasts to assess the relationship between Rb1, Cad11, and Cx43. Decreased RNA and protein expression of Cad11 were noted during differentiation of Rb1 deficient osteoblasts when compared to controls. Gap junctional intercellular communication was also studied and found to be reduced with Rb1 loss. Rb1 deficient cells had a higher steady-state level of adipocyte transcription factors and adipocyte markers when compared to control. Interestingly no changes were observed in Cx43 mRNA or protein expression when comparing the two lines. Immunofluorescence analysis demonstrated that a decrease in Cad11 may have affected its co-localization with Cx43. This, in turn, may have affected the proper formation of intercellular channels and thereby communication. These observations suggest that loss of Rb1 affects adhesion and communication in osteoblasts resulting in the expression of an adipocyte phenotype. Citation Format: Elisha Pendleton, Anthony Ketner, Thomas Bodenstine, Nalini Chandar. Role of retinoblastoma gene in maintenance of osteoblast function and communication [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2476.

Phthalate inhibits Leydig cell differentiation and promotes adipocyte differentiation

Studies have shown that phthalates are capable of affecting the development and functions of male reproductive system. The effect of phthalates on Leydig cell functions is well documented. However, little is known about their potential effects on the functions of stem Leydig cells (SLC). In the present study, we have examined the effects of mono-(2-ethylhexyl) phthalate (MEHP) on SLC functions in vitro by culturing seminiferous tubules and isolated SLCs. The results indicate that MEHP can significantly inhibit the proliferation and differentiation of SLCs in both the organ and cell culture systems. Interestingly, the minimal effective concentration that is able to affect SLC function was lower in the tubule culture system (1 μM) than in the isolated cells (10 μM), suggesting a possible involvement of the niche cells. Also, MEHP appeared to affect both the efficiency of SLCs to form Leydig cells and a selected group of Leydig cell-specific genes, including Lhcgr, Scarb1, Hsd3b1, Cyp17a1, Star, Srd5a1, Akr1c14, Insl3, Hao2 and Pah. Since SLCs are multipotent, we also tested the effect of MEHP on the differentiation of SLCs to adipocytes. Though MEHP by itself can not specify SLCs into adipocyte lineage, it indeed significantly increased the adipogenic activity of SLCs if used with an adipocyte inducing medium by up-regulation of multiple adipogenic-related genes, including Pparg and Cebpa. Overall, the results indicate that MEHP inhibits SLCs differentiating into Leydig lineage while stimulates the differentiating potential of SLCs to adipocytes.

Cynandione A induces adipogenesis and beige adipocyte-related phenotype in 3T3-L1 cells

Abstract Cynandione A (CA) is a major ingredient of Cynanchum wilfordii. Here, we report a new function of CA in adipogenesis in 3T3-L1 cells, a preadipocyte cell line. During adipogenic differentiation in 3T3-L1 cells, CA boosted the process by enhancing the expression of key adipogenic transcription factors (Pparg and C/ebpa), brown adipocyte-related genes (Prdm16, Pgc-1a, Cidea, and Ucp1), and beige adipocyte-related genes (Tbx1 and Cited1). Additionally, CA increased mitochondrial mass and expression levels of mitochondria-related genes (Sirt3 and Tfam) in the cells. These results suggest that CA induces adipogenesis and beige adipocyte-related phenotype in adipocyte lineage cells.

SRF-MRTF signaling suppresses brown adipocyte development by modulating TGF-β/BMP pathway

The SRF/MRTF and upstream signaling cascade play key roles in actin cytoskeleton organization and myocyte development. To date, how this signaling axis may function in brown adipocyte lineage commitment and maturation has not been delineated. Here we report that MRTF-SRF signaling exerts inhibitory actions on brown adipogenesis, and suppressing this negative regulation promotes brown adipocyte lineage development. During brown adipogenic differentiation, protein expressions of SRF, MRTFA/B and its transcription targets were down-regulated, and MRTFA/B shuttled from nucleus to cytoplasm. Silencing of SRF or MRTF-A/MRTF-B enhanced two distinct stages of brown adipocyte development, mesenchymal stem cell determination to brown adipocytes and terminal differentiation of brown adipogenic progenitors. We further demonstrate that the MRTF-SRF axis exerts transcriptional regulations of the TGF-β and BMP signaling pathway, critical developmental cues for brown adipocyte development. TGF-β signaling activity was significantly attenuated, whereas that of the BMP pathway augmented by inhibition of SRF or MRTF-A/MRTF-B, leading to enhanced brown adipocyte differentiation. Our study demonstrates the MRTF-SRF transcriptional cascade as a negative regulator of brown adipogenesis, through its transcriptional control of the TGF-β/BMP signaling pathways.

Gaucher disease-associated alterations in mesenchymal stem cells reduce osteogenesis and favour adipogenesis processes with concomitant increased osteoclastogenesis

Gaucher disease (GD) is caused by pathogenic mutations in GBA1, the gene that encodes the lysosomal enzyme β-glucocerebrosidase. Until now, treatments for GD cannot completely reverse bone problems. The aim of this work was to evaluate the potential of MSCs from GD patients (GD MSCs) to differentiate towards the osteoblast (GD Ob) and adipocyte (GD Ad) lineages, and their role in osteoclastogenesis. We observed that GD Ob exhibited reduced mineralization, collagen deposition and alkaline phosphatase activity (ALP), as well as decreased gene expression of RUNX2, COLA1 and ALP. We also evaluated the process of osteoclastogenesis and observed that conditioned media from GD MSCs supernatants induced an increase in the number of osteoclasts. In this model, osteoclastogenesis was induced by RANKL and IL-1β. Furthermore, results showed that in GD MSCs there was a promotion in NLRP3 and PPAR-γ gene expression. Adipogenic differentiation revealed that GD Ad had an increase in PPAR-γ and a reduced RUNX2 gene expression, promoting adipocyte differentiation. In conclusion, our results show that GD MSCs exhibited deficient GD Ob differentiation and increased adipogenesis. In addition, we show that GD MSCs promoted increased osteoclastogenesis through RANKL and IL-1β. These changes in GD MSCs are likely to contribute to skeletal imbalance observed in GD patients.

Abstract B24: A multicellular, microfluidic model of the human bone marrow niche in metastatic cancers

Abstract Bone metastases cause significant morbidity in solid tumors, with prostate, breast, lung, and kidney cancers representing the most common primary sources. Understanding the landscape of stromal interactions in bone and bone marrow would facilitate development of novel therapies targeting this unique metastatic niche. Mesenchymal stromal cells (MSCs) are multipotent cells in bone marrow that differentiate into osteoblasts, adipocytes, and fibroblasts and regulate immune and inflammatory responses. MSC recruitment into primary tumors enhances invasiveness of prostate adenocarcinoma, which suggests that bone marrow MSCs impact development of bone metastases in prostate cancer and promote treatment resistance. We hypothesize that multicellular interactions drive a permissive bone marrow environment for outgrowth of metastatic lesions. The goal of this study is to develop a multicellular bone stromal model from patients with solid tumors for functional and molecular analysis of cellular crosstalk. We have recently published a reconfigurable, microfluidic platform that enables culture of up to 6 different cell types in 3-dimensional matrices and multi-endpoint analysis of limited patient samples. In this study, we have modified this platform to culture patient-derived tissue, by using primary bone marrow cells from patients consented to provide bone marrow aspirate at time of radical prostatectomy. This technology allows spatiotemporal control of individual treatment conditions, enabling culture and differentiation of primary human cells in separate chips that can be reconfigured into defined microenvironments. Subsequently, specific compartments can be isolated for gene expression analysis and in-device imaging. Using this platform, we present two distinct bone marrow models. In the first model, we cultured primary human bone marrow MSCs that were induced into osteoblastic and adipocytic lineages in 3D microculture. MSCs induced invasion of prostate cancer cell lines, and paracrine crosstalk between MSCs and prostate cancer cells increased expression of IL-6, IL-8, CXCL12, and MMP-9 in MSCs. In the second model, we generated a multicellular mixed 3D culture in which osteoblasts and adipocytes spontaneously differentiate in coculture with stromal cells, osteoclasts, and leukocytes. The mixed 3D cultures generated spheroid structures that resemble stem cell components with self-renewal capability, previously called mesenspheres. Overall, we have recapitulated cellular compartments in the bone marrow niche using two complementary methods. The impact of defined molecular subtypes of cancer is now being tested to understand if specific genetic alterations promote development of invasive cancer in bone marrow. Future directions include analysis of paracrine signaling and functional interactions between disseminated cancer cells and bone marrow cellular stroma as potential therapeutic targets. Citation Format: Nan Sethakorn, David Kosoff, Erika Heninger, Jasmine Martinez Soto, Jiaquan Yu, Rahul Das, David Jarrard, Jacques Galipeau, Peiman Hematti, Scott Dehm, David J. Beebe, Joshua M. Lang. A multicellular, microfluidic model of the human bone marrow niche in metastatic cancers [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B24.

Syndecan-1 Facilitates the Human Mesenchymal Stem Cell Osteo-Adipogenic Balance.

Bone marrow-derived human mesenchymal stems cells (hMSCs) are precursors to adipocyte and osteoblast lineage cells. Dysregulation of the osteo-adipogenic balance has been implicated in pathological conditions involving bone loss. Heparan sulfate proteoglycans (HSPGs) such as cell membrane-bound syndecans (SDCs) and glypicans (GPCs) mediate hMSC lineage differentiation and with syndecan-1 (SDC-1) reported in both adipogenesis and osteogenesis, these macromolecules are potential regulators of the osteo-adipogenic balance. Here, we disrupted the HSPG profile in primary hMSC cultures via temporal knockdown (KD) of SDC-1 using RNA interference (RNAi) in undifferentiated, osteogenic and adipogenic differentiated hMSCs. SDC-1 KD cultures were examined for osteogenic and adipogenic lineage markers along with changes in HSPG profile and common signalling pathways implicated in hMSC lineage fate. Undifferentiated hMSC SDC-1 KD cultures exhibited a pro-adipogenic phenotype with subsequent osteogenic differentiation demonstrating enhanced maturation of osteoblasts. In cultures where SDC-1 KD was performed following initiation of differentiation, increased adipogenic gene and protein marker expression along with increased Oil Red O staining identified enhanced adipogenesis, with impaired osteogenesis also observed in these cultures. These findings implicate SDC-1 as a facilitator of the hMSC osteo-adipogenic balance during early induction of lineage differentiation.

The Proliferation and Differentiation of Adipose-Derived Stem Cells in Neovascularization and Angiogenesis.

Neovascularization and angiogenesis are vital processes in the repair of damaged tissue, creating new blood vessel networks and increasing oxygen and nutrient supply for regeneration. The importance of Adipose-derived Mesenchymal Stem Cells (ASCs) contained in the adipose tissue surrounding blood vessel networks to these processes remains unknown and the exact mechanisms responsible for directing adipogenic cell fate remain to be discovered. As adipose tissue contains a heterogenous population of partially differentiated cells of adipocyte lineage; tissue repair, angiogenesis and neovascularization may be closely linked to the function of ASCs in a complex relationship. This review aims to investigate the link between ASCs and angiogenesis/neovascularization, with references to current studies. The molecular mechanisms of these processes, as well as ASC differentiation and proliferation are described in detail. ASCs may differentiate into endothelial cells during neovascularization; however, recent clinical trials have suggested that ASCs may also stimulate angiogenesis and neovascularization indirectly through the release of paracrine factors.

Butein Promotes Lineage Commitment of Bone Marrow-Derived Stem Cells into Osteoblasts via Modulating ERK1/2 Signaling Pathways.

Butein is a phytochemical that belongs to the chalcone family of flavonoids and has antitumor, anti-inflammatory, and anti-osteoclastic bone resorption activities. This study aims to investigate the effects of butein on the differentiation potential of mouse primary bone marrow-derived mesenchymal stem cells (mBMSCs) into osteoblast and adipocyte lineages. Primary cultures of mBMSCs are treated with different doses of butein during its differentiation. Osteoblast differentiation is assessed by alkaline phosphatase (ALP) activity quantification and Alizarin red staining for matrix mineralization, while adipogenesis is assessed by quantification of lipid accumulation using Oil Red O staining. Osteoblastic and adipocytic gene expression markers are determined by quantitative real-time PCR (qPCR). Western blot analysis is used to study the activation of extracellular signal-regulated kinase (ERK1/2). Interestingly, butein promotes the lineage commitment of mBMSCs into osteoblasts, while suppressing their differentiation into adipocytes in a dose-dependent manner. A similar effect of butein is confirmed in human (h) primary BMSCs. Occurring at the molecular level, butein significantly upregulates the mRNA expression of osteoblast-related genes, while downregulating the expression of adipocyte-related genes. The mechanism of butein-induced osteogenesis is found to be mediated by activating the ERK1/2 signaling pathway. To conclude, we identify butein as a novel nutraceutical compound with an osteo-anabolic activity to promote the lineage commitment of BMSCs into osteoblast versus adipocyte. Thus, butein can be a plausible therapeutic drug for enhancing bone formation in osteoporotic patients.

The Molecular Anatomy of Mouse Skin during Hair Growth and Rest.

Skin homeostasis is orchestrated by dozens of cell types that together direct stem cell renewal, lineage commitment, and differentiation. Here, we use single-cell RNA sequencing and single-molecule RNA FISH to provide a systematic molecular atlas of full-thickness skin, determining gene expression profiles and spatial locations that define 56 cell types and states during hair growth and rest. These findings reveal how the outer root sheath (ORS) and inner hair follicle layers coordinate hair production. We found that the ORS is composed of two intermingling but transcriptionally distinct cell types with differing capacities for interactions with stromal cell types. Inner layer cells branch from transcriptionally uncommitted progenitors, and each lineage differentiation passes through an intermediate state. We also provide an online tool to explore this comprehensive skin cell atlas, including epithelial and stromal cells such as fibroblasts, vascular, and immune cells, to spur further discoveries in skin biology.

Stem Leydig Cells in the Adult Testis: Characterization, Regulation and Potential Applications.

Androgen deficiency (hypogonadism) affects males of all ages. Testosterone replacement therapy (TRT) is effective in restoring serum testosterone and relieving symptoms. TRT, however, is reported to have possible adverse effects in part because administered testosterone is not produced in response to the hypothalamic-pituitary-gonadal (HPG) axis. Progress in stem cell biology offers potential alternatives for treating hypogonadism. Adult Leydig cells (ALCs) are generated by stem Leydig cells (SLCs) during puberty. SLCs persist in the adult testis. Considerable progress has been made in the identification, isolation, expansion and differentiation of SLCs in vitro. In addition to forming ALCs, SLCs are multipotent, with the ability to give rise to all 3 major cell lineages of typical mesenchymal stem cells, including osteoblasts, adipocytes, and chondrocytes. Several regulatory factors, including Desert hedgehog and platelet-derived growth factor, have been reported to play key roles in the proliferation and differentiation of SLCs into the Leydig lineage. In addition, stem cells from several nonsteroidogenic sources, including embryonic stem cells, induced pluripotent stem cells, mature fibroblasts, and mesenchymal stem cells from bone marrow, adipose tissue, and umbilical cord have been transdifferentiated into Leydig-like cells under a variety of induction protocols. ALCs generated from SLCs in vitro, as well as Leydig-like cells, have been successfully transplanted into ALC-depleted animals, restoring serum testosterone levels under HPG control. However, important questions remain, including: How long will the transplanted cells continue to function? Which induction protocol is safest and most effective? For translational purposes, more work is needed with primate cells, especially human.

Comparison of the proteome patterns of adipose-derived stem cells with those treated with selegiline using a two dimensional gel electrophoresis analysis

ABSTRACTAdipose derived stem cells (ADSCs) are multipotent and can transdifferentiate into neural stem cells. We investigated the transdifferentiation of ADSCs to neural phenotype (NP) cells using selegiline and two-dimensional electrophoresis (2-DE). The perinephric and inguinal fat of rats was collected and used to isolate ADSCs that were characterized by immunophenotyping using flow cytometry. The ADSCs were differentiated into osteogenic and lipogenic cells. The NP cells were generated using 10−9 mM selegiline and characterized by immunocytochemical staining of nestin and neurofilament 68 (NF-68), and by qRT-PCR of nestin, neurod1 and NF68. Total protein of ADSCs and NP cells was extracted and their proteome patterns were examined using 2-DE. ADSCs carried CD73, CD44 and CD90 cell markers, but not CD34. ADSCs were differentiated into osteocyte and adipocyte lineages. The differentiated NP cells expressed nestin, neuro d1 and NF-68. The proteome pattern of ADSCs was compared with that of NP cells and eight spots showed more than a two fold increase in protein expression. The molecular weights and isoelectric points of these highly expressed proteins were estimated using Melanie software. We compared these results with those of the mouse proteomic database using the protein isoelectric point database, and the functions of the eight proteins in differentiation of NP cells were predicted using the UniProt database. The probable identities of the proteins that showed higher expression in NP cells included cholinesterase, GFRa2, protein kinase C (PKC-eta) and RING finger protein 121. The sequences of the proteins identified from mouse database were aligned by comparing them with similar proteins in rat database using the Basic Local Alignment Search Tool (BLAST). The E values of all aligned proteins were zero, which indicates consistency of the matched protein. These proteins participate in differentiation of the neuron and their overexpression causes ADSCs transdifferentiation into NP cells.