Ryan Berry

Adipocyte Lineage Cells Contribute to the Skin Stem Cell Niche to Drive Hair Cycling

In mammalian skin, multiple types of resident cells are required to create a functional tissue and support tissue homeostasis and regeneration. The cells that compose the epithelial stem cell niche for skin homeostasis and regeneration are not well defined. Here, we identify adipose precursor cells within the skin and demonstrate that their dynamic regeneration parallels the activation of skin stem cells. Functional analysis of adipocyte lineage cells in mice with defects in adipogenesis and in transplantation experiments revealed that intradermal adipocyte lineage cells are necessary and sufficient to drive follicular stem cell activation. Furthermore, we implicate PDGF expression by immature adipocyte cells in the regulation of follicular stem cell activity. These data highlight adipogenic cells as skin niche cells that positively regulate skin stem cell activity, and suggest that adipocyte lineage cells may alter epithelial stem cell function clinically.

Weighing in on adipocyte precursors.

Obesity, defined as an excessive increase in white adipose tissue (WAT), is a global health epidemic. In obesity, WAT expands by increased adipocyte size (hypertrophy) and number (hyperplasia). The location and cellular mechanisms of WAT expansion greatly affect the pathogenesis of obesity. However, the cellular and molecular mechanisms regulating adipocyte size, number, and depot-dependent expansion in vivo remain largely unknown. This perspective summarizes previous work addressing adipocyte number in development and obesity and discusses recent advances in the methodologies, genetic tools, and characterization of in vivo adipocyte precursor cells allowing for directed study of hyperplastic WAT growth in vivo.

Characterization of Cre recombinase models for the study of adipose tissue

The study of adipose tissue in vivo has been significantly advanced through the use of genetic mouse models. While the aP2-CreBI and aP2-CreSalk lines have been widely used to target adipose tissue, the specificity of these lines for adipocytes has recently been questioned. Here we characterize Cre recombinase activity in multiple cell populations of the major adipose tissue depots of these and other Cre lines using the membrane-Tomato/membrane-GFP (mT/mG) dual fluorescent reporter. We find that the aP2-CreBI and aP2-CreSalk lines lack specificity for adipocytes within adipose tissue, and that the aP2-CreBI line does not efficiently target adipocytes in white adipose depots. Alternatively, the Adiponectin-CreERT line shows high efficiency and specificity for adipocytes, while the PdgfRα-CreERUCL and PdgfRα-CreERJHU lines do not efficiently target adipocyte precursor cells in the major adipose depots. Instead, we show that the PdgfRα-Cre line is preferable for studies targeting adipocyte precursor cells in vivo.

A High Fat Diet Increases Bone Marrow Adipose Tissue (MAT) But Does Not Alter Trabecular or Cortical Bone Mass in C57BL/6J Mice.

Obesity has been associated with high bone mineral density (BMD) but a greater propensity to fracture. Some obese individuals have increased marrow adipose tissue (MAT), but the impact of MAT on bone turnover remains controversial, as do changes in BMD associated with a high fat diet (HFD). In this study we hypothesized that MAT volume would increase in response to HFD but would be independent of changes in BMD. Hence, we fed C57BL/6J (B6) male mice at 3 weeks of age either a high fat diet (60 kcal %) or regular diet (10 kcal %) for 12 weeks (n = 10/group). We measured MAT volume by osmium staining and micro‐CT (µCT) as well as bone parameters by µCT, histomorphometry, and dual‐energy X‐ray absorptiometry. We also performed a short‐term pilot study using 13‐week‐old B6 males and females fed a HFD (58 kcal %) for 2 weeks (n = 3/sex). Both long‐ and short‐term HFD feedings were associated with high MAT volume, however, femoral trabecular bone volume fraction (BV/TV), bone formation rate and cortical bone mass were not altered in the long‐term study. In the short‐term pilot study, areal BMD was unchanged after 2 weeks of HFD. We conclude that, for B6 mice fed a HFD starting at wean or 13 weeks of age, MAT increases whereas bone mass is not altered. More studies are needed to define the mechanism responsible for the rapid storage of energy in the marrow and its distinction from other adipose depots. J. Cell. Physiol. 230: 2032–2037, 2015.

The Adipose Tissue Microenvironment Regulates Depot-Specific Adipogenesis in Obesity

The sexually dimorphic distribution of adipose tissue influences the development of obesity-associated pathologies. The accumulation of visceral white adipose tissue (VWAT) that occurs in males is detrimental to metabolic health, while accumulation of subcutaneous adipose tissue (SWAT) seen in females may be protective. Here, we show that adipocyte hyperplasia contributes directly to the differential fat distribution between the sexes. In male mice, high-fat diet (HFD) induces adipogenesis specifically in VWAT, while in females HFD induces adipogenesis in both VWAT and SWAT in a sex hormone-dependent manner. We also show that the activation of adipocyte precursors (APs), which drives adipocyte hyperplasia in obesity, is regulated by the adipose depot microenvironment and not by cell-intrinsic mechanisms. These findings indicate that APs are plastic cells, which respond to both local and systemic signals that influence their differentiation potential independent of depot origin. Therefore, depot-specific AP niches coordinate adipose tissue growth and distribution.

Use of osmium tetroxide staining with microcomputerized tomography to visualize and quantify bone marrow adipose tissue in vivo.

Adipocytes reside in discrete, well-defined depots throughout the body. In addition to mature adipocytes, white adipose tissue depots are composed of many cell types, including macrophages, endothelial cells, fibroblasts, and stromal cells, which together are referred to as the stromal vascular fraction (SVF). The SVF also contains adipocyte progenitors that give rise to mature adipocytes in those depots. Marrow adipose tissue (MAT) or marrow fat has long been known to be present in bone marrow (BM) but its origin, development, and function remain largely unknown. Clinically, increased MAT is associated with age, metabolic diseases, drug treatment, and marrow recovery in children receiving radiation and chemotherapy. In contrast to the other depots, MAT is unevenly distributed in the BM of long bones. Conventional quantitation relies on sectioning of the bone to overcome issues with distribution but is time-consuming, resource intensive, inconsistent between laboratories and may be unreliable as it may miss changes in MAT volume. Thus, the inability to quantitate MAT in a rapid, systematic, and reproducible manner has hampered a full understanding of its development and function. In this chapter, we describe a new technique that couples histochemical staining of lipid using osmium tetroxide with microcomputerized tomography to visualize and quantitate MAT within the medullary canal in three dimensions. Imaging of osmium staining provides a high-resolution map of existing and developing MAT in the BM. Because this method is simple, reproducible, and quantitative, we expect it will become a useful tool for the precise characterization of MAT.

Determination of mesenchymal stem cell fate by pigment epithelium-derived factor (PEDF) results in increased adiposity and reduced bone mineral content

Pigment epithelium‐derived factor (PEDF), the protein product of the SERPINF1 gene, has been linked to distinct diseases involving adipose or bone tissue, the metabolic syndrome, and osteogenesis imperfecta (OI) type VI. Since mesenchymal stem cell (MSC) differentiation into adipocytes vs. osteoblasts can be regulated by specific factors, PEDF‐directed dependency of murine and human MSCs was assessed. PEDF inhibited adipogenesis and promoted osteoblast differentiation of murine MSCs, osteoblast precursors, and human MSCs. Blockade of adipogenesis by PEDF suppressed peroxisome proliferator‐activated receptor‐γ (PPARγ), adiponectin, and other adipocyte markers by nearly 90% compared with control‐treated cells (P<0.001). Differentiation to osteoblasts by PEDF resulted in a common pathway that involved PPARγ suppression (P<0.01). Canonical Wnt‐β‐catenin signaling results in a MSC differentiation pattern analogous to that seen with PEDF. Thus, adding PEDF enhanced Wnt‐β‐catenin signal transduction in human MSCs, demonstrating a novel Wnt agonist function. In PEDF knockout (KO) mice, total body adiposity was increased by >50% compared with controls, illustrating its systemic role as a negative regulator of adipogenesis. Bones from KO mice demonstrated a reduction in mineral content recapitulating the OI type VI phenotype. These results demonstrate that the human diseases associated with PEDF reflect its ability to modulate MSC differentiation.—Gattu, A. K., Swenson, E. S., Iwakiri, Y., Samuel, V. T., Troiano, N., Berry, R., Church, C. D., Rodeheffer, M. S., Carpenter, T. O., Chung, C. Determination of mesenchymal stem cell fate by pigment epithelium‐derived factor (PEDF) results in increased adiposity and reduced bone mineral content. FASEB J. 27, 4384–4394 (2013). www.fasebj.org

Isolation and Study of Adipocyte Precursors

White adipose tissue (WAT) is a heterogeneous tissue composed of lipid-filled adipocytes and several nonadipocyte cell populations, including endothelial, blood, uncharacterized stromal, and adipocyte precursor cells. Although lipid-filled adipocytes account for the majority of WAT volume and mass, nonadipocyte cell populations have critical roles in WAT maintenance, growth, and function. As mature adipocytes are terminally differentiated postmitotic cells, differentiation of adipocyte precursors is required for hyperplastic WAT growth during development and in obesity. In this chapter, we present methods to separate adipocyte precursor cells from other nonadipocyte cell populations within WAT for analysis by flow cytometry or purification by fluorescence-activated cell sorting. Additionally, we provide methods to study the adipogenic capacity of purified adipocyte precursor cells ex vivo.

Imaging of adipose tissue.

Adipose tissue is an endocrine organ that specializes in lipid metabolism and is distributed throughout the body in distinct white adipose tissue (WAT) and brown adipose tissue (BAT) depots. These tissues have opposing roles in lipid metabolism with WAT storing excessive caloric intake in the form of lipid, and BAT burning lipid through nonshivering thermogenesis. As accumulation of lipid in mature adipocytes of WAT leads to obesity and increased risk of comorbidity (Pi-Sunyer et al., 1998), detailed understanding of the mechanisms of BAT activation and WAT accumulation could produce therapeutic strategies for combatting metabolic pathologies. As morphological changes accompany alterations in adipose function, imaging of adipose tissue is one of the most important tools for understanding how adipose tissue mass fluctuates in response to various physiological contexts. Therefore, this chapter details several methods of processing and imaging adipose tissue, including bright-field colorimetric imaging of paraffin-sectioned adipose tissue with a detailed protocol for automated adipocyte size analysis; fluorescent imaging of paraffin and frozen-sectioned adipose tissue; and confocal fluorescent microscopy of whole mounted adipose tissue. We have also provided many example images showing results produced using each protocol, as well as commentary on the strengths and limitations of each approach.

Adipose Tissue Residing Progenitors (Adipocyte Lineage Progenitors and Adipose Derived Stem Cells (ADSC).

The formation of brown, white, and beige adipocytes has been a subject of intense scientific interest in recent years due to the growing obesity epidemic in the USA and around the world. This interest has led to the identification and characterization of specific tissue-resident progenitor cells that give rise to each adipocyte population in vivo. However, much still remains to be discovered about each progenitor population in terms of their “niche” within each tissue and how they are regulated at the cellular and molecular level during healthy and diseased states. While our knowledge of brown, white, and beige adipose tissue is rapidly increasing, little is still known about marrow adipose tissue and its progenitor despite recent studies demonstrating possible roles for marrow adipose tissue in regulating the hematopoietic space and systemic metabolism at large. This chapter focuses on our current knowledge of brown, white, beige, and marrow adipose tissue with a specific focus on the formation of each tissue from tissue-resident progenitor cells.