Stromal Vascular Cells Research Articles

Essential role of Ahnak in adipocyte differentiation leading to the transcriptional regulation of Bmpr1\u03b1 expression

The role of Ahnak in obesity has been reported previously. Loss of Ahnak leads to decreased Bmp4/Smad1 signaling, resulting in the downregulation of adipocyte differentiation. However, the biological significance of Ahnak remains largely unknown. In this study, we demonstrate that Ahnak-mediated impaired adipogenesis results in decreased Bmpr1α transcriptional expression. To confirm this, Ahnak siRNA was used to knock-down Ahnak in C3H10T1/2 and primary stromal vascular fraction cells. Ahnak siRNA transfected cells showed suppression of Bmpr1α expression and decreased BMP4/ Bmpr1α signaling. The differential adipogenesis was further confirmed by knock-down of Bmpr1α in C3H10T1/2 cells, which resulted in reduced adipogenesis. Moreover, stable Ahnak knock-out C3H10T1/2 cells stably transfected with Ahnak CRISPR/Cas9 plasmid suppressed expression of Bmpr1α and prevented differentiation into adipocytes. Furthermore, we developed immortalized pre-adipocytes from wild-type or Ahnak Knock-out mice’s stromal vascular fraction (SVF) to confirm the function of Ahnak in pre-adipocyte transition. Immortalized Ahnak knock-out SVF cells showed lower level of Bmpr1α expression, evidence by their impaired BMP4/Bmpr1α signaling. Upon adipogenic induction, immortalized Ahnak knock-out SVF cells exhibited a marked decrease in adipocyte differentiation compared with immortalized wild-type pre-adipocytes. Furthermore, over-expression of Bmpr1α restored the adipogenic activity of Ahnak knock-out C3H10T1/2 cells and immortalized Ahnak knock-out SVF cells. Our data reveal the missing link in Ahnak-mediated adipose tissue remodeling and suggest that precise regulation of Ahnak in adipose tissue might have a therapeutic advantage for metabolic disease treatment.

Development of xeno-free epithelial differentiation media for adherent, non-expanded adipose stromal vascular cell cultures

IntroductionReconstruction of respiratory epithelium is critical for the fabrication of bioengineered airway implants. Epithelial differentiation is typically achieved using bovine pituitary extract (BPE). Due to the xenogenic nature and undefined composition of BPE, an alternative for human clinical applications, devoid of BPE, must be developed. The goal of this study was to develop two different BPE-free media, with and without select pituitary hormone (PH), which could initiate epithelial differentiation for use in human implantation. MethodsThe ability of the two BPE-free media to initiate epithelial differentiation of adherent, non-expanded stromal-vascular cells grown on porcine small intestinal submucosa was compared to traditional BPE-containing media (M1). Nanostring® was used to measure differences in gene expression of stemness (MSC), basal cell (basal), and ciliated markers (muco-cil), and staining was performed support the gene data. ResultsCompared to baseline, both BPE-free media upregulated epithelial and stemness genes, however this was to a lower degree than BPE-containing media. In general, the expression of basal cell markers (COL17A1, DSG3, ITGA6, KRT6A, LOXL2) and secreted mucous proteins (PLUNC, MUC5B, SCGB2A1) was upregulated. The gene expression of ciliated markers C9orf24, TUBA3 and DNCL2B but not of the key transcription factor for cilagenesis FOXJ1 were upregulated, indicating that mucus-secreting cell differentiation occurs more rapidly than ciliogenesis. The ability of the adherent stromal vascular cells to upregulate gene expression of both epithelial and stemness markers suggests maintenance of the self-renewal capacity of undifferentiated and/or basal cell-like cells contributing to proliferation and ensuring a persisting source of cells for regenerative medicine applications. ConclusionThis study provides the initial step to defining a BPE-free epithelial differentiation medium for clinical translation. Thus, either of the proposed BPE-free medium are viable alternatives to BPE-containing medium for partial epithelial differentiation for human translational applications.

Isolation and characterization of minipig perivascular stem cells for bone tissue engineering.

Human subcutaneous adipose tissue has been recognized as a rich source of tissue resident mesenchymal stem/stromal cells (MSC) in recent years. The current study was designed to sort the minipig (mp) perivascular stem cells (PSCs) and investigate the osteogenic potential. Purification of human PSCs was achieved via fluorescence-activated cell sorting (FACS) from human liposuction samples [cluster of differentiation (CD)45−CD34−CD146+ perithelial cells and CD45−CD34+CD146− adventitial cells]. Subsequently, PSCs were isolated from mp adipose tissue samples (n=9), characterized and, using purified mpPSCs (obtained by FACS, which is used in human PSC purification), the mpPSC osteogenic and adipogenic potential was evaluated by Alizarin Red S and Oil Red O staining in vitro, respectively. The cell morphometry was observed following cell isolation and culture, and hematoxylin and eosin staining was performed to identify the fat tissue structure and vascular distribution. Osteogenic and adipogenic differentiation-associated gene expression levels were analyzed by reverse transcription-quantitative polymerase chain reaction. The results demonstrated that the same antigens used for human PSC identification and isolation were working in mp tissue (CD45, CD146 and CD34). The two cell groups: CD45−CD34−CD146+ pericytes and CD45−CD34+CD146− adventitial cells were successfully isolated from the subcutaneous fat in the posterior neck of mps, mpPSCs accounted for 8.6% of the stromal vascular fraction (SVF) with 1.4% pericytes and 7.2% adventitial cells. mpPSCs demonstrated characteristics of MSCs, including cell surface marker expression, colony forming unit-fibroblast inclusion, and the stronger osteogenic and adipogenic differentiation potential than that of the non-selected vascular stromal cells. The mRNA expression levels of osteocalcin, collagen, type I, α1 and peroxisome proliferator-activated receptor-γ in the mpPSCs group were significantly higher than those of the unsorted pSVF group (P<0.05). Thus, the current study successfully isolated and cultured CD146+ and CD34+ cell populations from mp tissues, characterized the cells' PSC-like phenotype and identified their distinctly osteogenic and adipogenic potential.

Distinct Bone Marrow Sources of Pleiotrophin Control Hematopoietic Stem Cell Maintenance and Regeneration.

Bone marrow (BM) perivascular stromal cells and vascular endothelial cells (ECs) are essential for hematopoietic stem cell (HSC) maintenance, but the roles of distinct niche compartments during HSC regeneration are less understood. Here we show that Leptin receptor-expressing (LepR+) BM stromal cells and ECs dichotomously regulate HSC maintenance and regeneration via secretion of pleiotrophin (PTN). BM stromal cells are the key source of PTN during steady-state hematopoiesis because its deletion from stromal cells, but nothematopoietic cells, osteoblasts, or ECs, depletes the HSC pool. Following myelosuppressiveirradiation, PTN expression is increased in bone marrow endothelial cells (BMECs), and PTN+ECs are more frequent in the niche. Moreover, deleting Ptn from ECs impairs HSC regeneration whereas Ptn deletion from BM stromal cells does not. These findings reveal dichotomous and complementary regulation of HSC maintenance and regeneration by BM stromal cells and ECs.

Omentin1 gene expression in epicardial and subcutaneous adipose tissue during obesity related coronary heart disease

Aim: Adipose tissue is an endocrine organ synthesizes a number of biologically active compounds known as adipocytokines that regulate glucose and lipid homeostasis. Imbalance in adipokines production and secretion by specialized adipose tissue types can be linked to metabolic and cardiovascular diseases. Due to its anatomic and functional proximity to the myocardium epicardial fat activity can be most important in the development of coronary heart disease (CHD). Omentin-1 is an anti-inflammatory adipokine expressed in stromal vascular cells of the adipose tissue and suggested to play a role in obesity, diabetes and CHD progression. Our hypothesis was that Omentin 1 gene (ITLN1) expression variations in epicardial adipose tissue can be associated with obesity-related CHD.

Targetted inhibition of CD74 attenuates adipose COX-2-MIF-mediated M1 macrophage polarization and retards obesity-related adipose tissue inflammation and insulin resistance.

Adipose tissue (AT) inflammation is crucial to the development of obesity-associated insulin resistance. Our aim was to investigate the contribution of cyclooxygenase-2 (COX-2)/macrophage migration inhibitory factor (MIF)-mediated cross-talk between hypertrophic adipocytes and macrophages to the etiology of AT inflammation and the involvement of CD74 using human SGBS adipocytes, THP-1 macrophages and mice fed a high-fat (HF) diet. The MIF and CD74 mRNA levels in the adipocytes and stromal vascular cells (SVCs) of white fat were highly correlated with body weight (BW), homeostatic model assessment for insulin resistance (HOMA-IR), and adipose macrophage marker expression levels, especially those in SVCs. COX-2 inhibition suppressed the elevation of MIF production in HF white adipocytes as well as palmitate and hypoxic-treated SGBS adipocytes. Treatment of adipocytes transfected with shCOX-2 and siMIF or subjected to MIF depletion in the medium reversed the pro-inflammatory responses in co-incubated THP-1 cells. Inhibition of NF-κB activation reversed the COX2-dependent MIF secretion from treated adipocytes. The targetted inhibition of macrophage CD74 prevented M1 macrophage polarization in the above co-culture model.The COX-2-dependent increases in CD74 gene expression and MIF release in M1-polarized macrophages facilitated the expression of COX-2 and MIF in co-cultured SGBS adipocytes. CD74 shRNA intravenous injection suppressed HF-induced AT M1 macrophage polarization and inflammation as well as insulin resistance in mice. The present study suggested thatCOX-2-mediated MIF secretion through NF-κB activation from hypertrophic and hypoxic adipocytes as well as M1 macrophages might substantially contribute to the phenotypic switch of AT macrophages through CD74 in obesity. Inhibition of CD74 could attenuate AT inflammation and insulin resistance in the development of HF diet-induced obesity.

TGF-β receptor 1 regulates progenitors that promote browning of white fat

ObjectiveBeige/brite adipose tissue displays morphological characteristics and beneficial metabolic traits of brown adipose tissue. Previously, we showed that TGF-β signaling regulates the browning of white adipose tissue. Here, we inquired whether TGF-β signals regulated presumptive beige progenitors in white fat and investigated the TGF-β regulated mechanisms involved in beige adipogenesis. MethodsWe deleted TGF-β receptor 1 (TβRI) in adipose tissue (TβRIAdKO mice) and, using flow-cytometry based assays, identified and isolated presumptive beige progenitors located in the stromal vascular cells of white fat. These cells were molecularly characterized to examine beige/brown marker expression and to investigate TGF-β dependent mechanisms. Further, the cells were transplanted into athymic nude mice to examine their adipogenesis potential. ResultsDeletion of TβRI promotes beige adipogenesis while reducing the detrimental effects of high fat diet feeding. Interaction of TGF-β signaling with the prostaglandin pathway regulated the appearance of beige adipocytes in white fat. Using flow cytometry techniques and stromal vascular fraction from white fat, we isolated presumptive beige stem/progenitor cells (iBSCs). Upon genetic or pharmacologic inhibition of TGF-β signaling, these cells express high levels of predominantly beige markers. Transplantation of TβRI-deficient stromal vascular cells or iBSCs into athymic nude mice followed by high fat diet feeding and stimulation of β-adrenergic signaling via CL316,243 injection or cold exposure promoted robust beige adipogenesis in vivo. ConclusionsTβRI signals target the prostaglandin network to regulate presumptive beige progenitors in white fat capable of developing into beige adipocytes with functional attributes. Controlled inhibition of TβRI signaling and concomitant PGE2 stimulation has the potential to promote beige adipogenesis and improve metabolism.

Cartilage Regeneration in Humans with Adipose Tissue-Derived Stem Cells and Adipose Stromal Vascular Fraction Cells: Updated Status.

Adipose tissue-derived stem cells (ASCs) in the form of stromal vascular fraction (SVF) and cultured expansion have been applied in clinical settings in some countries to treat osteoarthritis (OA) of knees, one of the most common debilitating, incurable disorders. Since the first report of successful cartilage-like tissue regeneration with autologous adipose SVF containing ASCs, there has been a gradual increase in the number of publications confirming such results. Thus far, most of the reports have been limited to treatments of OA of knees. Recently, successful applications of adipose SVF in treating OA of ankles and hips have been reported. In addition, several groups have reported modified methods of applying adipose SVF, such as combining bone marrow stimulation with adipose SVF or adding additional extracellular matrix (ECM) in treating OA. Here, we present an updated, systematic review of clinical effectiveness and safety in treating OA of knees, ankles, and one hip since 2016 using ASCs in the form of adipose SVF or in cultured expansion, along with a description and suggestion of potential biological mechanisms of cartilage regeneration.

Therapeutic Efficacy of Adipose-Derived Stromal Vascular Fraction Cells is associated with CD34 Positivity in Acute-on-Chronic Liver Failure

Background Adipose-derived stromal vascular fraction (SVF) cells, a rich source of primary stem/stromal cells, are promising for administering cell therapy for patients with acute-on-chronic liver failure (ACLF). We evaluated the therapeutic effects of CD34+/CD34− SVF cells in hepatocyte cotransplantation in a rat model of ACLF. Method ACLF was induced in Sprague–Dawley rats by temporary bile duct ligation (chronic) and D-galactosamine administration (acute). Donor hepatocytes and SVF cells were isolated from Tg(UBC–emGFP) rats and human adipose tissues, respectively. Sorted CD34+ and CD34− SVF cells were separated using a magnetic bead system. Rat hepatocytes and SVF cells (labeled with the PKH26 fluorescent dye) were intraportally transplanted into ACLF rats. The surviving animals were sacrificed at 1 and 2 weeks after the transplantation. Results ACLF was evidenced by the onset of acute coagulopathy and hepatocyte necrosis in fibrotic livers. Compared with the control group, the unsorted group showed less biliary ductular proliferation and fibrosis at 1 week after the transplantation. Furthermore, prominent biliary ductular proliferation and significantly progression of fibrosis (P = .005) were observed in the CD34− group than in the CD34+ group. At 2 weeks, the serum level of alkaline phosphatase was significantly higher in the CD34− group than in the CD34+ group (P = .007). The transplanted SVF cells were found in the periportal regions at 1 week, whereas donor hepatocytes were not detected and proliferation of endogenous hepatocytes was observed at times. Conclusion Compared with CD34− SVF cells, cotransplantation of CD34+ SVF cells resulted in the early amelioration of liver fibrosis and biliary ductular proliferation. Heterogeneous therapeutic effects of SVF cells were associated with CD34 positivity, which poses translational significance in future cell therapy.

PO-292 Breast cancer stem cell reprogramming: deciphering the impact of glucose and the contribute of tumour microenvironment

IntroductionDiabetes-associated hyperglycemia is linked to poorer prognosis and survival in breast cancer (BC). Indeed, glucose can affect both tumour and tumour-surrounding cells. BC cells are embedded in an adipocyte-rich microenvironment, which, beside adipocytes, contains Stromal-Vascular Fraction Cells (SVFCs). In this scenario, epithelial and stromal compartments communicate through the release of soluble factors and establish an intricate crosstalk. Here, we analysed whether glucose could directly affect the phenotype of ER+ MCF7 BC cells and interfere with their interaction with adipose-derived SVFCs, thereby promoting tumour progression.Material and methodsMCF7 cell stemness markers were measured by qReal-Time PCR. Adipose-derived (Ad-)SVFCs were obtained by mammary adipose tissue specimens of women undergoing plastic surgery. The trascriptome of MCF7 exposed to either low (LG-5.5 mM) or high glucose concentration (HG-25 mM) was obtained by RNA-Sequencing (Illumina HiSeq3000).Results and discussionsHG exposure of MCF7 determined a significant increase of SOX2 mRNA levels as compared to LG, suggesting the induction of stemness programming. Co-culture with Ad-SVFCs in HG increased SOX2, NANOG and OCT4 mRNA levels in MCF7, as compared to isolated culture, indicating the involvement of SVF-produced soluble factors in BC stem cell reprogramming. Moreover, in presence of Ad-SVFCs and HG, MCF7 produced a higher number of mammospheres, which also displayed larger size. However, both in LG and in HG, conditioned media (CM) obtained from Ad-SVFCs produced no relevant effect on MCF7 stemness. Nevertheless, when Ad-SVFCs were pre-incubated with CM obtained from HG-treated MCF7, their CM very effectively increased OCT4, NANOG and SOX2 mRNA levels in MCF7. Thus, HG likely perturbs MCF7, which produce soluble factors leading Ad-SVFCs to release, in turn, reprogramming factors for BC cell stemness. In this regard, we have observed that HG modification of MCF7 transcriptome includes deregulation of 17 genes (pval=0.05) encoding for secreted proteins involved in cancer progression-related pathways, which may potentially play a role in tumour-stroma interactions.ConclusionGlucose affects BC stem cell reprogramming both directly and through Ad-SVFCs. Deciphering the mechanisms that govern this intricate crosstalk will pave the way to new targeted strategies to improve BC control in conditions of metabolic derangement.

Small Proliferative Adipocytes, Newly Identified Adipose Progenitor, as the Precursors of Beige Cells

We previously identified proliferative cells in adipose tissue expressing adipocyte specific genes and preadipocyte specific genes, which were named small proliferative adipocytes (SPA). Furthermore, we found that SPA specifically express proliferin (PLF) and have superior potential for adipogenic differentiation as compared to stromal vascular cells (SVC). In this study, we investigated the role of SPA. A comparison between differentiated SPA (D-SPA) and differentiated SVC (D-SVC) revealed that UCP1 and PLF were strongly expressed in D-SPA. Therefore, we speculated that SPA might be progenitor of beige cells. The real time PCR analyses and the immunohistochemical studies revealed that beige cells associated genes, including UCP1, PPARα and PGC1α were significantly increased in D-SPA. Treatment with pioglitazone and CL316234, a specific β3-adrenergic receptor agonist, converted cultured SPA into cell clusters like beige cells which expressed UCP1 and activation of mitochondrial function confirmed by MitoRed stain. Furthermore, PLF was expressed in beige cells induced by continuous injection of CL316243. In conclusion, since D-SPA were found to express UCP1 and PLF, which were detected in beige cells, but not WAT, SPA were considered to be a progenitor of beige cells. There might be two lineages of cells in adipose tissue; adipose progenitor cells in SVC and WAT (PLF-), and SPA and beige cells (PLF+). The combination of pioglitazone and CL316423 produced an additive effect of inducing browning in SPA. Disclosure K. Taguchi: None. K. Kajita: None. M. Fuwa: None. M. Asano: None. Y. Kitada: None. T. Ikeda: None. T. Ishizuka: None. H. Morita: None.

Obesity-Associated Family with Sequence Similarity 13 Member A Gene Regulates Adipocyte Differentiation

Obesity and its associated morbidities represent the major and most rapidly expanding health epidemic in the world. Recent genome-wide association studies (GWAS) have identified single nucleotide polymorphism (SNP) variants in Family with Sequence Similarity 13, Member A (FAM13A) gene to be highly associated with waist-hip ratio (WHR) with adjustment for body mass index (BMI) (WHRadjBMI). However, the exact function of this gene in adipose tissue development and obesity remains largely uncharacterized. We for the first time identified Fam13A as an adipose tissue abundant gene whose expression was highly regulated by fasting/refeed and dramatically reduced in epididymal and subcutaneous fat depots in murine models of genetic obesity and diet-induced obesity (DIO). Its expression was enriched in mature adipocytes and significantly upregulated during murine and human adipogenesis potentially through a PPARγ-dependent mechanism. However, FAM13A-/- mice only exhibited a tendency of higher adiposity and maintained normal glucose clearance and insulin resistance. They are also not protected from high fat diet-induced obesity and metabolic perturbation. The dispensable role of FAM13A was further supported by normal adipogenesis in FAM13A-/- Stromal vascular cells (SVCs). Interestingly, overexpression of FAM13A in 3T3-L1 preadipocytes downregulated beta-catenin expression, impaired preadipocyte survival and enhanced its susceptibility to serum starvation-induced apoptosis. Furthermore, FAM13A-overexpressing preadipocytes failed to differentiate into mature adipocytes through interfering early-stage C/EBPα/PPARγ-mediated adipogenesis. These data for the first time reveal new details of an obesity-associated FAM13A gene in mediating beta-catenin action and adipocyte differentiation, providing novel insight into a relationship between FAM13a and adipose biology. Disclosure J. Tang: None. H. Zhou: None. J. Yang: None. W. Chen: None.

Siah2 Expression in Adipocyte Progenitor Cells

Obesity-related insulin resistance is associated with adipose tissue inflammation and visceral adipose tissue expansion via adipocyte hypertrophy and impaired recruitment of new progenitor cells to undergo adipogenesis (hyperplasia). Deletion of the ubiquitin ligase Seven-in-absentia homolog 2 (Siah2) in obese mice significantly reduced adipose tissue inflammation and insulin resistance compared to obese wild type mice. However, larger adipocyte size accompanied this phenotype and subsequent experiments with adipose tissue stromal vascular fraction (SVF) cells showed adipogenesis required Siah2. In an earlier study, we found Siah2 affects adipogenesis by enhancing gene expression of the PPARγ coactivator, Zfp423 via proteasome-dependent degradation of ZFP521, a transcriptional repressor of Zfp423. ZFP423 is an early marker of preadipocyte determination that is expressed in adipocyte progenitor cells. Although Siah2 mediates adipogenesis and is highly expressed in the adipose tissue stromal vascular cell fraction, cell-specific expression of Siah2 in adipose tissue is not well characterized. We found that Siah2 is highly expressed in macrophage. RNA-seq and microarray data from the Immunological Genome Project (Immgen.org) also reported Siah2 expression in lymphocytes. To identify Siah2 expression profile in adipose tissue, unsorted cells and magnetically sorted macrophage, T-cell, B-cell, and SCA-1 positive progenitor cells from mouse adipose tissues SVF were analyzed for Siah2 expression. In parallel, in situ hybridization RNAscope of fat pads was employed to inspect Siah2 mRNA localization in adipose tissue. Since our evidence indicates Siah2 acts upstream of ZFP423, we predict Siah2 is expressed in adipocyte progenitor cells as well as immune cells in adipose tissue. This work will provide the groundwork to better understand how Siah2 controls commitment of progenitor cells to the adipocyte lineage and links obesity to adipose tissue inflammation and insulin resistance. Disclosure T. Dang: None. G.E. Kilroy: None. J.L. Taylor: None. Y. Yu: None. E. Floyd: None.

Single-Cell RNA Sequencing Identifies Functionally Distinct Fibro-inflammatory and Adipogenic Pdgfrr Progenitor Subpopulations in Visceral Adipose Tissue

White adipose tissue (WAT) remodeling is dictated by coordinated interactions between adipocytes and resident stromal-vascular cells; however, the functional heterogeneity of adipose stromal cells has remained unresolved. We combined single-cell RNA-sequencing and FACS to identify and isolate functionally distinct subpopulations of Pdgfrβ stromal cells within visceral WAT of adult mice. Ly6c- Cd9- Pdgfrβ cells represent highly adipogenic visceral adipocyte precursor cells (“APCs”), whereas Ly6c Pdgfrβ cells represent fibro-inflammatory progenitors (“FIPs”). FIPs lack adipogenic capacity, display pro-fibrogenic/proinflammatory phenotypes, and can exert an anti-adipogenic effect on APCs. The pro-inflammatory and adipogenic phenotypes of Pdgfrβ cells are regulated, at least in part, by Nr4a nuclear receptors. These data highlight the functional heterogeneity of visceral WAT perivascular cells, and provide insight into potential cell-cell interactions impacting adipogenesis and inflammation. These improved strategies to isolate FIPs and APCs from visceral WAT will facilitate the study of physiological WAT remodeling and mechanisms leading to metabolic dysfunction.

A novel role for PTK2B in cultured beige adipocyte differentiation

Adipocyte differentiation is a tightly regulated process which requires the sequential and organized expression of numerous genes and proteins. Phosphorylation of cytoplasmic proteins and key transcription factors represents a critical regulatory mechanism of the process leading to adipocyte maturation and modulation of associated metabolic pathways. Despite the recognition of the importance of protein phosphorylation in adipocyte biology, relatively little is known about the role of specific kinases in thermogenic (brown or beige) adipocyte differentiation and function. In this study, we demonstrate that the non-receptor protein tyrosine kinase 2 beta (PTK2B) plays a critical role in murine beige adipocyte differentiation. We observed that PTK2B protein expression is associated with beige adipocyte differentiation in cultured, immortalized, inguinal stromal vascular fraction cells. CRISPR/Cas9-mediated knock-out of Ptk2b results in non-differentiating white adipocytes, and differentiated beige adipocytes with significantly reduced thermogenic gene and protein expression, enlarged lipid droplet size, and altered mitochondrial respiration. Together, our data in a cell culture system provides evidence for a role of PTK2B in the differentiation of murine beige adipocytes.

Cellular therapy with human autologous adipose-derived adult cells of stromal vascular fraction for alopecia areata

BackgroundMost common forms of hair loss (alopecia) are caused by aberrant hair follicle cycling and changes in hair follicle morphology. However, current treatments for alopecia do not specifically target these processes. Adipose-derived stromal vascular cells (ADSVCs) that can be harvested from fat cells are one of the latest breakthroughs in the aesthetic field. The potential use of stem cell-based therapies (SCBT) for the repair and regeneration of various tissues and organs offers a paradigm shift that may provide alternative therapeutic solutions, which can be applied to prevent hair loss. This study aimed to present clinical cases of SCBT for the treatment of alopecia areata by transplantation of ADSVCs in the scalp.MethodsTwenty patients (9 women and 11 men) were recruited to our retrospectively registered study. After lipoaspiration, autologous ADSVCs were generated and characterized before the injection of 4–4.7 × 106 cells into the scalp of the patient. Hair regeneration was assessed by three clinical tests: the pull test, hair quality, and hair density.ResultsAll patients experienced hair regeneration, increased hair growth and decreased pull test 3 and 6 months after the treatment with ADSVCs [hair density (85.1 ± 8.7 vs 121.1 ± 12.5 hair/cm2, P < 0.0001), hair diameter (60.5 ± 1.8 vs 80.8 ± 2.4μ, P < 0.0001) and pull-test values (4.4 ± 0.3 vs 0.8 ± 0.2, P < 0.0001), untreated versus 6 months post-operative)]. Significant variation was observed between men and women only for hair diameter. No significant differences were observed with age.ConclusionsThe obtained results prove the efficacy and the safety of the treatment, and satisfaction of the patients confirm the quality of the results.

Photobiomodulation of freshly isolated human adipose tissue-derived stromal vascular fraction cells by pulsed light-emitting diodes for direct clinical application.

A highly interesting source for adult stem cells is adipose tissue, from which the stromal vascular fraction (SVF)-a heterogeneous cell population including the adipose-derived stromal/stem cells-can be obtained. To enhance the regenerative potential of freshly isolated SVF cells, low-level light therapy (LLLT) was used. The effects of pulsed blue (475nm), green (516nm), and red (635nm) light from light-emitting diodes applied on freshly isolated SVF were analysed regarding cell phenotype, cell number, viability, adenosine triphosphate content, cytotoxicity, and proliferation but also osteogenic, adipogenic, and proangiogenic differentiation potential. The colony-forming unit fibroblast assay revealed a significantly increased colony size after LLLT with red light compared with untreated cells, whereas the frequency of colony-forming cells was not affected. LLLT with green and red light resulted in a stronger capacity to form vascular tubes by SVF when cultured within 3D fibrin matrices compared with untreated cells, which was corroborated by increased number and length of the single tubes and a significantly higher concentration of vascular endothelial growth factor. Our study showed beneficial effects after LLLT on the vascularization potential and proliferation capacity of SVF cells. Therefore, LLLT using pulsed light-emitting diode light might represent a new approach for activation of freshly isolated SVF cells for direct clinical application.

Regenerative Potential and Inflammation-Induced Secretion Profile of Human Adipose-Derived Stromal Vascular Cells Are Influenced by Donor Variability and Prior Breast Cancer Diagnosis.

Adipose tissue contains a heterogeneous population of stromal vascular fraction (SVF) cells that work synergistically with resident cell types to enhance tissue healing. Ease of access and processing paired with therapeutic promise make SVF cells an attractive option for autologous applications in regenerative medicine. However, inherent variability in SVF cell therapeutic potential from one patient to another hinders prognosis determination for any one person. This study investigated the regenerative properties and inflammation responses of thirteen, medically diverse human donors. Using non-expanded primary lipoaspirate samples, SVF cells were assessed for robustness of several parameters integral to tissue regeneration, including yield, viability, self-renewal capacity, proliferation, differentiation potential, and immunomodulatory cytokine secretion. Each parameter was selected either for its role in regenerative potential, defined here as the ability to heal tissues through stem cell repopulation and subsequent multipotent differentiation, or for its potential role in wound healing through trophic immunomodulatory activity. These data were then analyzed for consistent and predictable patterns between and across measurements, while also investigating the influence of the donors' relevant medical histories, particularly if the donor was in remission following breast cancer treatment. Analyses identified positive correlations among the expression of three cytokines: interleukin (IL)-6, IL-8, and monocyte chemoattractant protein (MCP)-1. The expression of these cytokines also positively related to self-renewal capacity. These results are potentially relevant for establishing expectations in both preclinical experiments and targeted clinical treatment strategies that use stem cells from patients with diverse medical histories.

Berberine hydrochloride induces brown adipogenesis

Brown adipose tissue (BAT) dissipates energy in the form of heat through uncoupled respiration. Brown adipocytes originate from different precursor cells and can be induced by various stimuli. Pharmacologically induction of BAT thermogenesis has been shown to be a promising treatment for obesity‐related metabolic disease. In this study, we investigated the potential role of berberine hydrochloride (BBR), a naturally occurring plant alkaloid, in activating brown fat adipogenesis.3T3L1 preadipocyte cells, C2C12 myoblasts, and stromal vascular cells isolated from perigonadal fat of C57BL/6 mice were cultured in cell differentiation induction media for three days followed by differentiation maintenance media for another three days in the presence or absence of 10 μM of BBR. C2C12 differentiation media was also supplemented with PPARγ agonist rosiglitazone (0.5μM). Adipocyte differentiation was confirmed by oil red staining. Western blot analysis was performed on the differentiated cells to measure brown fat specific protein markers including UCP1, PCG‐1α, PPARγ, PRDM16 and CIDE‐A.BBR treatment significantly induced all of the above brown‐fat specific markers (p&lt;0.005; n=4) in 3T3L1 cells, C2C12 myoblasts and stroma vascular blast cells. Specifically, UCP‐1 expression is increased 30.3 fold in 3T3L‐1, and 2.00 fold in primary adipocytes extracted from WAT respectively.Our results thus indicate that BBR induces brown adipogenesis, and is a promising treatment for obesity and obesity related metabolic diseases.Support or Funding InformationStart Up fund (Central Michigan University )This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Human Body Composition and Immunity: Visceral Adipose Tissue Produces IL-15 and Muscle Strength Inversely Correlates with NK Cell Function in Elderly Humans.

Natural killer (NK) lymphocyte-mediated cytotoxicity and cytokine secretion control infections and cancers, but these crucial activities decline with age. NK cell development, homeostasis, and function require IL-15 and its chaperone, IL-15 receptor alpha (IL-15Rα). Macrophages and dendritic cells (DC) are major sources of these proteins. We had previously postulated that additional IL-15 and IL-15Rα is made by skeletal muscle and adipose tissue. These sources may be important in aging, when IL-15-producing immune cells decline. NK cells circulate through adipose tissue, where they may be exposed to local IL-15. The objectives of this work were to determine (1) if human muscle, subcutaneous adipose tissue (SAT), and visceral adipose tissue (VAT) are sources of IL-15 and IL-15 Rα, and (2) whether any of these tissues correlate with NK cell activity in elderly humans. We first investigated IL-15 and IL-15Rα RNA expression in paired muscle and SAT biopsies from healthy human subjects. Both tissues expressed these transcripts, but IL-15Rα RNA levels were higher in SAT than in skeletal muscle. We also investigated tissue obtained from surgeries and found that SAT and VAT expressed equivalent amounts of IL-15 and IL-15Rα RNA, respectively. Furthermore, stromal vascular fraction cells expressed more IL-15 RNA than did adipocytes. To test if these findings related to circulating IL-15 protein and NK cell function, we tested 50 healthy adults aged > 70 years old. Plasma IL-15 levels significantly correlated with abdominal VAT mass in the entire cohort and in non-obese subjects. However, plasma IL-15 levels did not correlate with skeletal muscle cross-sectional area and correlated inversely with muscle strength. Plasma IL-15 did correlate with NK cell cytotoxic granule exocytosis and with CCL4 (MIP-1β) production in response to NKp46-crosslinking. Additionally, NK cell responses to K562 leukemia cells correlated inversely with muscle strength. With aging, immune function declines while infections, cancers, and deaths increase. We propose that VAT-derived IL-15 and IL-15Rα is a compensatory NK cell support mechanism in elderly humans.