Precise Intradermal Injection of Nanofat-Derived Stromal Cells Combined with Platelet-Rich Fibrin Improves the Efficacy of Facial Skin Rejuvenation.

BACKGROUND/AIMS: The rejuvenation properties of nanofat grafting have been described in recent years. However, it is not clear whether the clinical efficacy of the procedure is attributable to stem cells or linked to other components of adipose tissue. In this study we isolated nanofat-derived stem cells (NFSCs) to observe their biological characteristics and evaluate the efficacy of precise intradermal injection of nanofat combined with platelet-rich fibrin (PRF) in patients undergoing facial rejuvenation treatment. METHODS: Third-passage NFSCs were isolated and cultured using a mechanical emulsification method and their surface CD markers were analyzed by flow cytometry. The adipogenic and osteogenic nature and chondrogenic differentiation capacity of NFSCs were determined using Oil Red O staining, alizarin red staining, and Alcian blue staining, respectively. Paracrine function of NFSCs was evaluated by enzyme-linked immunosorbent assay (ELISA) at 1, 3, 7, 14, and 28 days after establishing the culture. Then, the effects of PRF on NFSC proliferation were assessed in vitro. Finally, we compared the outcome in 103 patients with facial skin aging who underwent both nanofat and intradermal PRF injection (treatment group) and 128 patients who underwent hyaluronic acid (HA) injection treatment (control group). Outcomes in the two groups were compared by assessing pictures taken at the same angle before and after treatment, postoperative recovery, incidence of local absorption and cysts, and skin quality before treatment, and at 1, 12, 24 months after treatment using the VISIA Skin Image Analyzer and a SOFT5.5 skin test instrument. RESULTS: NFSCs expressed CD29, CD44, CD49d, CD73, CD90, and CD105, but did not express CD34, CD45, and CD106. NFSCs also differentiated into adipocytes, osteoblasts, and chondrocytes under appropriate induction conditions. NFSCs released large amounts of growth factors such as VEGF, bFGF, EGF, and others, and growth factor levels increased in a time-dependent manner. At the same time, PRF enhanced proliferation of NFSCs in vitro in a dose-dependent manner, and the growth curves under different concentrations of PRF all showed plateaus 6d after seeding. Facial skin texture was improved to a greater extent after combined injection of nanofat and PRF than after control injection of HA. The nanofat-PRF group had a higher satisfaction rate. Neither treatment caused any complications such as infection, anaphylaxis, or paresthesia during long-term follow-up. CONCLUSION: NFSCs demonstrate excellent multipotential differentiation and paracrine function, and PRF promotes proliferation of NFSCs during the early stage after seeding. Both nanofat-PRF and HA injection improve facial skin status without serious complications, but the former was associated with greater patient satisfaction, implying that nanofat-PRF injection is a safe, highly effective, and long-lasting method for skin rejuvenation.

Characterization of Chondrogenic Gene Expression and Cartilage Phenotype Differentiation in Human Breast Adipose-Derived Stem Cells Promoted by Ginsenoside Rg1 In Vitro.

BACKGROUND/AIMS: Investigating and understanding chondrogenic gene expression during the differentiation of human breast adipose-derived stem cells (HBASCs) into chondrogenic cells is a prerequisite for the application of this approach for cartilage repair and regeneration. In this study, we aim to characterize HBASCs and to examine chondrogenic gene expression in chondrogenic inductive culture medium containing ginsenoside Rg1. METHODS: Human breast adipose-derived stem cells at passage 3 were evaluated based on specific cell markers and their multilineage differentiation capacity. Cultured HBASCs were treated either with basic chondrogenic inductive conditioned medium alone (group A, control) or with basic chondrogenic inductive medium plus 10 µg/ml (group B), 50 µg/ml (group C), or 100µg/ml ginsenoside Rg1 (group D). Cell proliferation was assessed using the CCK-8 assay for a period of 9 days. Two weeks after induction, the expression of chondrogenic genes (collagen type II, collagen type XI, ACP, COMP and ELASTIN) was determined using real-time PCR in all groups. RESULTS: The different concentrations of ginsenoside Rg1 that were added to the basic chondrogenic inductive culture medium promoted the proliferation of HBASCs at earlier stages (groups B, C, and D) but resulted in chondrogenic phenotype differentiation and higher mRNA expression of collagen type II (CO-II), collagen type XI (CO-XI), acid phosphatase (ACP), cartilage oligomeric matrix protein (COMP) and ELASTIN compared with the control (group A) at later stages. The results reveal an obvious positive dose-effect relationship between ginsenoside Rg1 and the proliferation and chondrogenic phenotype differentiation of HBASCs in vitro. CONCLUSIONS: Human breast adipose-derived stem cells retain stem cell characteristics after expansion in culture through passage 3 and serve as a feasible source of cells for cartilage regeneration in vitro. Chondrogenesis in HBASCs was found to be prominent after chondrogenic induction in conditions containing ginsenoside Rg1.

Human breast adipose-derived stem cells transfected with the stromal cell-derived factor-1 receptor CXCR4 exhibit enhanced viability in human autologous free fat grafts.

BACKGROUND: The main complication of autologous free fat tissue transplantation is fat resorption and calcification due to the ischemic necrosis of fat. The promotion of transplant neovascularization soon after autologous free fat grafts may reduce these outcomes. In adulthood, stromal cell-derived factor-1 (SDF-1) and its membrane receptor C-X-C chemokine receptor type 4 (CXCR4) are involved in the homing and migration of multiple stem cell types, neovascularization, and cell proliferation. We hypothesized that CXCR4 may improve the long-term survival of free fat tissue transplants by recruiting endothelial progenitor cells (EPCs) and may therefore improve graft revascularization. In this study, we aimed to determine the effect of human breast adipose-derived stem cells (HBASCs) transfected with the CXCR4 gene on the survival rate of human autologous free fat transplants in nude mice. METHODS: Human breast adipose-derived stem cells (HBASCs) were expanded ex vivo for 3 passages, labeled with green fluorescent protein (GFP) and transfected with CXCR4 or left untransfected. Autologous fat tissues were mixed with the GFP-labeled, CXCR4-transfected HBASCs (group A), GFP-labeled HBASCs (group B), the known vascularization-promoting agent VEGF (group C), or medium (group D) and then injected subcutaneously into 32 nude mice at 4 spots in a random fashion. Six months later, the transplanted tissue volume and histology were evaluated, and neo-vascularization was quantified by counting the capillaries. CXCR4 and SDF-1α mRNA expression in the transplants was determined using real-time quantitative PCR analysis (qPCR). RESULTS: The data revealed that the control (group D) transplant volume survival was 28.3 ± 4.5%. Mixing CXCR4-transfected (group A) and untransfected (group B) HBASCs significantly increased transplant volume survival (79.5 ± 8.3% and 67.2 ± 5.9%, respectively), whereas VEGF-transfected HBASCs (group C) were less effective (41.2 ± 5.1%). Histological analysis revealed that both types of HBASCs-treated transplants consisted predominantly of adipose tissue, unlike the control transplants, and also presented significantly less fat necrosis and fibrosis. The CXCR4-transfected HBASCs-treated transplants had a significantly higher capillary density than did the other transplants and showed GFP and CD31 double-positive cells (i.e., ASCs-derived endothelial cells). The mRNA expression of CXCR4 and SDF-1α was much higher in the CXCR4-transfected HBASCs transplants than in the other three transplants. CONCLUSIONS: Our data demonstrated that HBASCs can enhance the survival and quality of transplanted free fat tissues. Moreover, CXCR4 transfection of these HBASCs could augment this effect. Stimulation of angiogenesis and decreased fat cell apoptosis due to the recruitment of endothelial progenitor cells (EPCs) and an increase in graft revascularization are potential mechanisms underlying the improved long-term survival of free fat transplants following CXCR4-transfected HBASCs treatment.

Effect of ginsenoside Rg1 on proliferation and neural phenotype differentiation of human adipose-derived stem cells in vitro.

AIMS: To investigate whether ginsenoside Rg1 can promote neural phenotype differentiation of human adipose-derived stem cells (hASCs) in vitro. METHODS: hASCs were isolated from lipo-aspirates, and characterized by specific cell markers and multilineage differentiation capacity after culturing to the 3rd passage. Cultured hASCs were treated with neural inductive media alone (group A, control) or inductive media plus 10, 50, or 100 µg/mL ginsenoside Rg1 (groups B, C, and D, respectively). Cell proliferation was assessed by CCK-8 assay. Neuron specific enolase (NSE) and microtubule-associated protein-2 (MAP-2) levels were measured by Western blot. mRNA levels of growth associated protein-43 (GAP-43), neural cell adhesion molecule (NCAM), and synapsin-1 (SYN-1) were determined by real-time PCR. RESULTS: Ginsenoside Rg1 promoted the proliferation of hASCs (groups B, C, and D) and resulted in higher expression of NSE and MAP-2 compared with the control group. Gene expression levels of GAP-43, NCAM, and SYN-1 in the test groups were higher than that in thw control. The results displayed a dose-dependent effect of ginsenoside Rg1 on cell proliferation and neural phenotype differentiation. CONCLUSION: This study indicated that ginsenoside Rg1 promotes cell proliferation and neural phenotype differentiation of hASCs in vitro, suggesting a potential use for hASCs in neural regeneration medicine.

Advanced Platelet-Rich Fibrin Extract Treatment Promotes the Proliferation and Differentiation of Human Adipose-Derived Mesenchymal Stem Cells through Activation of Tryptophan Metabolism.

BACKGROUND: Advanced platelet-rich fibrin extract (APRFE) contains a high concentration of various cytokines that are helpful for improving stem cells repair function. OBJECTIVE: However, the underlying mechanism of APRFE improving stem cell repairing is not clear. METHODS: We produced APRFE by centrifuging fresh peripheral blood samples and isolated and identified human adipose-derived mesenchymal stem cells (ADMSCs). The abundance of cytokines contained in APRFE was detected by the Enzyme-linked immunosorbent assay (ELISA). The ADMSCs treated with or without APRFE were collected for transcriptome sequencing. RESULTS: Based on the sequencing data, the expression profiles were contracted. The differentially expressed genes and lncRNA (DEGs and DElncRNAs) were obtained using for the differential expression analysis. The lncRNA-miRNA-mRNA network was constructed based on the miRNet database. The further enrichment analysis results showed that the biological functions were mainly related to proliferation, differentiation, and cell-cell function. To explore the role of APRFE, the protein-protein interaction network was constructed among the cytokines included in APRFE and DEGs. Furthermore, we constructed the global regulatory network based on the RNAInter and TRRUST database. The pathways in the global regulatory network were considered as the core pathways. We found that the DEGs in the core pathways were associated with stemness scores. CONCLUSION: In summary, we predicted that APRFE activated three pathways (tryptophan metabolism, mTOR signaling pathway, and adipocytokine signaling) to promote the proliferation and differentiation of ADMSCs. The finding may be helpful for guiding the application of ADMSCs in the clinic.

The Effects of Hedgehog Signaling Pathway on the Proliferation and Apoptosis of Melanoma Cells.

BACKGROUND: Studies have found that the abnormality of the Hedgehog signaling pathway is related to the occurrence and development of a variety of tumors, but the effect of this signaling pathway on melanoma cells is still unclear. METHODS: This study aimed to discuss the effect of Hedgehog signaling pathway on the proliferation and apoptosis of human malignant melanoma A375 cells and explore its possible mechanism in the proliferation and apoptosis of melanoma cells. Different concentrations of Hedgehog signaling pathway inhibitor cyclopamine (5, 10, 20 and 40 µM) were used to treat human melanoma A375 cells for 24, 48, and 72 h, and set a blank control group (0 µM). Trypan blue cell counting method was used to detect cell viability. MTT method was used to detect the inhibition rate of cell proliferation. Transwell was used to detect cell invasion, and flow cytometry was used to detect cell apoptosis. RESULTS: Through the trypan blue cell counting method and MTT experiment, it was found that the Hedgehog signaling pathway inhibitor cyclopamine has an inhibitory effect on the proliferation and viability of melanoma A375 cells (P < 0.05), and the proliferation inhibitory effect is enhanced with prolonged action time in a dose- and time-dependent manner. Transwell experiment showed that compared with the blank control group, the invasion and migration ability of the treated melanoma A375 cells are significantly reduced, and the difference is statistically significant (P < 0.05). Cell apoptosis experiment showed that compared with the blank control group, the apoptosis rate of A375 cells is significantly higher after treated by 40 µM cyclopamine for 24 h, and the difference is statistically significant (P < 0.05). Gli1 and Bcl-2 protein are highly expressed in melanoma A375 cells, and their expressions show a downward trend (P < 0.05) after being treated by cyclopamine. CONCLUSION: Cyclopamine inhibits cell proliferation and induces cell apoptosis by downregulating Gli1. Hedgehog signaling pathway can be used as a new target for the treatment of malignant melanoma, and multiple measures can be used to inhibit the signaling pathway to achieve a therapeutic effect.

Rg1 Promotes the Proliferation and Adipogenic Differentiation of Human Adipose-Derived Stem Cells via FXR1/Lnc-GAS5-AS1 Pathway.

BACKGROUND: Human adipose-derived stem cells (hASCs) play an important role in regenerative medicine. OBJECTIVE: Exploring the mechanism of Rg1 in the promotion of the proliferation and adipogenic differentiation of hASCs is important in regenerative medicine research. METHODS: To observe ginsenoside Rg1 in promoting the proliferation and adipogenic differentiation of hASCs, Rg1 medium at different concentrations was established and tested using the cell counting kit-8 (CCK-8) assay, oil red O staining, alizarin red, and alcian blue. Compared to the control, differentially expressed genes (DEGs) were screened via DEG analysis, which was carried out in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. To explore the relationship among mRNA, long non-coding RNA (lncRNA) and microRNA (miRNA), we constructed a competing endogenous RNA (ceRNA) network. RESULTS: In this study, Rg1 was observed to promote the proliferation and adipogenic differentiation of hASCs. Additionally, enriched BPs and KEGG pathways may be involved in the promotion process, where FXR1 and Lnc-GAS5-AS1 were found to be regulatory factors. The regulatory network suggested that Rg1 could regulate the adipocytokine signaling pathway and IL-17 signaling pathway via FXR1 and Lnc-GAS5-AS1, which served as the mechanism encompassing the promotion of Rg1 on the proliferation and adipogenic differentiation of hASCs. CONCLUSION: A comprehensive transcriptional regulatory network related to the promotion ability of Rg1 was constructed, revealing mechanisms regarding Rg1's promotion of the proliferation and adipogenic differentiation of hASCs. The present study provides a theoretical basis for optimizing the function of hASCs.

Resveratrol Mediates the Apoptosis of Triple Negative Breast Cancer Cells by Reducing POLD1 Expression.

Resveratrol (RSV) is known to possess anticancer properties in many types of cancers like breast cancer, in which POLD1 may serve as a potential target. However, the anticancer mechanism of RSV on triple negative breast cancer (TNBC) remains unclear. In the present study, the antitumor effects and mechanism of RSV on TNBC cells were analyzed by RNA sequencing (RNA-seq), which was then verified via cell counting kit-8 (CCK8), immunofluorescence, immunohistochemistry, Western Blot (WB), flow cytometry, and hematoxylin-eosin (HE) staining. According to the corresponding findings, the survival rate of MDA-MB-231 cells gradually decreased as RSV treatment concentration increased. The RNA-seq analysis results demonstrated that genes affected by RSV treatment were mainly involved in apoptosis and the p53 signaling pathway. Moreover, apoptosis of MDA-MB-231 cells induced by RSV was observed to be mainly mediated by POLD1. When treated with RSV, the expression levels of full length PARP1, PCNA, and BCL-2 were found to be significantly reduced, and the expression level of Cleaved-PARP1 as well as Cleaved-Caspase3 increased significantly. Additionally, the mRNA expression of POLD1 was significantly reduced after treatment with RSV, and the protein expression level was also inhibited by RSV in a concentration-dependent manner. The prediction of domain interaction suggested that RSV may bind to at least five functional domains of the POLD1 protein (6s1m, 6s1n, 6s1o, 6tny and 6tnz). Furthermore, after RSV treatment, the anti-apoptotic index (PCNA, BCL-2) of MDA-MB-231 cells was found to decrease while the apoptosis index (caspase3) increased. Moreover, the overexpression of POLD1 reduced the extent of apoptosis observed in MDA-MB-231 cells following RSV treatment. Moreover, animal experimental results showed that RSV had a significant inhibitory effect on the growth of live tumors, while POLD1 overexpression was shown to antagonize this inhibitory effect. Accordingly, this study's findings reveal that RSV may promote the apoptosis of TNBC cells by reducing the expression of POLD1 to activate the apoptotic pathway, which may serve as a potential therapy for the treatment of TNBC.

Liquid-Liquid Phase Separation-Related Genes Associated with Tumor Grade and Prognosis in Hepatocellular Carcinoma: A Bioinformatic Study.

AIM: The aim of the present study was to identify the association between tumor grade and liquid-liquid phase separation (LLPS)-related genes, and to generate a LLPS-related gene-based risk index (LLPSRI) as a prognostic tool for hepatocellular carcinoma (HCC). METHODS: Weighted gene correlation network analysis was performed to test whether the LLPS-related gene modules were associated with tumor grade of HCC. The candidate modules were subjected to functional enrichment analysis. We generated a LLPSRI using the expression profiles of the hub genes among the candidate modules in order to identify patients at high risk. Then, the biological characteristics of the high-risk patients were revealed using gene set enrichment analysis. Additionally, an independent external data set was used to validate the LLPSRI. RESULTS: Four gene modules showed a significant positive correlation with tumor grade and involved various cancer-related pathways. Among the hub genes, six were selected to generate the LLPSRI, which was significantly associated with prognosis of HCC patients. The LLPSRI could successfully divide patients with HCC into high- and low-risk groups, and patients in the high-risk group showed shorter overall survival than those in the low-risk group. E2F, MYC, and mTORC1 signaling may be important determinants of survival in the high-risk group. The prognostic value of the LLPSRI was validated with the independent external data set. CONCLUSION: We identified LLPS-related gene modules that are associated with HCC tumor grade. The LLPSRI may be useful as a prognostic marker of HCC, and it may reliably stratify patients into groups at low or high risk of worse survival. Our analysis also suggests that certain biological characteristics of HCC may be associated with high risk of worse survival.

FGF2-induced PI3K/Akt signaling evokes greater proliferation and adipogenic differentiation of human adipose stem cells from breast than from abdomen or thigh.

In this study, human adipose stem cells were isolated from subcutaneous fat in the thigh (htASCs), abdomen (haASCs) and breast (hbASCs). Flow cytometry was used to detect cell surface markers, and an enzyme-linked immunosorbent assay was used to detect paracrine activity. Paracrine gene expression in the three cell types was examined using real-time qPCR, and adipogenic ability was assessed using Oil Red O staining. RNA from third-passage haASCs and hbASCs was sequenced. The results showed that the differentiation potential marker markers CD49d and CD54 were similar across hbASCs from 10 subjects. The hbASCs showed higher colony forming ability and expression of fibroblast growth factor-2, tissue inhibitor of metalloproteinase-1 and stromal cell derived factor-1 than htASCs and haASCs. Stimulating hbASCs with FGF2 promoted adipogenic differentiation, while treating the cells with the PI3K inhibitor LY294002 inhibited differentiation. These results suggest that the PI3K/Akt signaling pathway can promote proliferation and adipogenic differentiation of adipose stem cells, and that activation of this pathway by FGF2 may explain why hbASCs show greater proliferation and adipogenic differentiation than haASCs and htASCs.

Exploring the mechanism of resistance to sorafenib in two hepatocellular carcinoma cell lines.

Sorafenib has long been the only approved systemic therapy for advanced hepatocellular carcinoma (HCC), but most patients show primary or acquired drug resistance. In the present study, RNA was extracted from sorafenib-resistant and -sensitive clones of the HCC cell lines HepG2 and Huh7. Protein-protein interaction networks of the up- and down-regulated genes common to the two sorafenib-resistant cell lines were extracted and subjected to modular analysis in order to identify functional modules. Functional enrichment analysis showed the modules were involved in different biological processes and pathways. These results indicate that sorafenib resistance in HCC is complicated and heterogeneous. The potential regulators of each functional module, including transcription factors, microRNAs and long non-coding RNAs, were explored to construct a comprehensive transcriptional regulatory network related to sorafenib resistance in HCC. Our results provide new insights into sorafenib resistance of HCC at the level of transcriptional regulation.

Multiomics global landscape of stemness-related gene clusters in adipose-derived mesenchymal stem cells.

BACKGROUND: Adipose-derived mesenchymal stem cells (AD-MSCs) are a type of stem cell that is abundant and widely used. The molecular characteristics of AD-MSCs from different passages from donors of different ages have not been well elucidated. METHODS: Six kinds of AD-MSCs ((E1, E2, E3, Y1, Y2, and Y3) with E denoting cells derived from an elderly patient, Y denoting cells derived from a young patient, and 1, 2, and 3 representing passages 3, 6, and 10) were obtained from human abdominal adipose tissue. We obtained the protein expression profile, the mRNA expression profile, the lncRNA expression profile, and the methylation profile of each kind of AD-MSC by sequencing. After calculating the stemness indices, genes related to stemness were extracted. The multiomics correlation analysis was performed in the stemness-related genes. In addition, short time-series expression miner (STEM) analysis was performed for all cell passages and donor ages. To further explore the biological functions of the stemness-related genes, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Finally, the lncRNA-KEGG network and transcription factor (TF)-KEGG network were constructed based on the RNAInter database and TRRUST v2 database. RESULTS: The stemness of the Y1, E1, and Y2 cells was higher than that of the E2, Y3, and E3 cells. The stemness was the highest for Y1 cells and the lowest for E3 cells. STEM analysis showed that five stemness-related gene clusters were associated with the cell passages, and only one gene cluster was associated with age. The enrichment analysis results showed that the biological processes (BPs) and KEGG pathways were mainly involved in the proliferation, differentiation, and migration of cells. The global regulatory landscape of AD-MSCs was constructed: 25 TFs and 16 lncRNAs regulated 21 KEGG pathways through 27 mRNAs. Furthermore, we obtained a core stemness-related gene set consisting of ITGAV, MAD2L1, and PCNA. These genes were expressed at higher levels in Y1 cells than in E3 cells. CONCLUSION: The multiomics global landscape of stemness-related gene clusters was determined for AD-MSCs, which may be helpful for selecting AD-MSCs with increased stemness.

Landscape of transcription and expression regulated by DNA methylation related to age of donor and cell passage in adipose-derived mesenchymal stem cells.

Adipose-derived mesenchymal stem cells (ADSCs) are pluripotent stromal cells that can differentiate into a variety of cell types, including skin cells. High-throughput sequencing was performed on cells of different ages and cell passage, obtaining their methylation, mRNA expression, and protein profile data. The stemness of each sample was then calculated using the TCGAbiolinks package in R. Co-expression modules were identified using WGCNA, and a crosstalk analysis was performed on the corresponding modules. The ClusterProfile package was used for the functional annotation of module genes. Finally, the regulatory network diagram was visualized using the Cytoscape software. First, a total of 16 modules were identified, where 3 modules were screened that were most relevant to the phenotype. 29 genes were screened in combination of the RNA seq, DNA methylation seq and protein iTRAQ. Finally, a comprehensive landscape comprised of RNA expression, DNA methylation and protein profiles of age relevant ADSCs was constructed. Overall, the different omics of ADSCs were comprehensively analyzed in order to reveal mechanisms pertaining to their growth and development. The effects of age, cell passage, and stemness on the therapeutic effect of ADSCs were explored. Additionally, a theoretical basis for selecting appropriate ADSC donors for regenerative medicine was provided.

Ginsenoside Rg1 Accelerates Paracrine Activity and Adipogenic Differentiation of Human Breast Adipose-Derived Stem Cells in a Dose-Dependent Manner In Vitro.

Augmenting the biological function of adipose-derived stromal cells (ASCs) is a promising approach to promoting tissue remodeling in regenerative medicine. Here, we examined the effect of ginsenoside Rg1 on the paracrine activity and adipogenic differentiation capacity of human breast ASCs (hbASCs) in vitro. hbASCs were isolated and characterized in terms of stromal cell surface markers and multipotency. Third-passage hbASCs were cultured in basic media only or basic media containing different concentrations of G-Rg1 (0.1-100 µM). Cell proliferation was assessed by CCK-8 assay. Paracrine activity was assessed using ELISA. Gene expression was measured by qRT-PCR. Adipogenic differentiation capacity was evaluated by Oil red O staining. We found that hbASCs differentiated into adipocytes, osteoblasts, and chondrocytes in appropriate induction culture medium. hbASCs showed expression of CD29, CD44, CD49d, CD73, CD90, CD105, and CD133 but not CD31 and CD45 surface markers. G-Rg1 increased hbASC proliferation and adipogenic differentiation capacity at lower concentrations (0.1-1 µM) and had the opposite effects at higher concentrations (10-100 µM), while enhanced paracrine activity was observed in all experimental groups compared with control group, and the activation effect of lower concentration G-Rg1 was greater than at higher concentration. These results indicate that G-Rg1 can enhance the proliferation, paracrine activity, and adipogenic differentiation capacity of hbASCs within a certain concentration range. Therefore, the use of G-Rg1 may be beneficial to ASC-assisted fat graft regeneration and soft tissue engineering.

[Design of myocutaneous flap with color Doppler flow imaging technique for breast reconstruction in breast cancer after operation and radiotherapy].

OBJECTIVE: To investigate the method and effect of axial pattern myocutaneous flap in reconstructing breast by using color doppler flow imaging (CDFI) technique. METHODS: Suitable axial myocutaneous flaps were selected according to the character of the focus in 26 cases of breast cancer after operation and radiotherapy. All the axial pattern myocutaneous flaps were designed on the basis of traditional design method before operation; then, CDFI with high resolution was used to examine the starting spot, exterior diameter, trail and length of the myocutaneous flaps' major artery. The myocutaneous flaps were redesigned according to the results of CDFI and transferred to reconstruct the breasts. The results of operation and examination were investigated. RESULTS: According to the CDFI, only one thoracodorsal artery's blood current was slow, its wall was rough and presented with arteriosclerosis. The blood flow was fluent and the vessel wall was smooth with other supplying arteries in the flaps. And no embolism, sclerosis or absence of blood vessel was found. The starting spots, exterior diameters, trails and anatomic layers of the major supplying arteries of the flaps were displayed clearly with CDFI, in accordance with the results of operation. Twenty-one cases of latissimus dorsi myocutaneous flap, 4 cases of the contralateral transverse abdominis myocutaneous flap and 1 cases of the bilateral transverse abdominis myocutaneous flap were used in this group. The flaps survived and healed well, the breasts were reconstructed well with perfect appearance, shape and sensation. CONCLUSIONS: CDFI is a simple, visualized and noninvasive method for designing the axial pattern myocutaneous flap in breast reconstruction, it can provide more scientific and accurate evidence for preoperative determination of myocutaneous flap transplantation.

Nanofat-derived stem cells with platelet-rich fibrin improve facial contour remodeling and skin rejuvenation after autologous structural fat transplantation.

Traditional autologous fat transplantation is a common surgical procedure for treating facial soft tissue depression and skin aging. However, the transplanted fat is easily absorbed, reducing the long-term efficacy of the procedure. Here, we examined the efficacy of nanofat-assisted autologous fat structural transplantation. Nanofat-derived stem cells (NFSCs) were isolated, mechanically emulsified, cultured, and characterized. Platelet-rich fibrin (PRF) enhanced proliferation and adipogenic differentiation of NFSCs in vitro. We then compared 62 test group patients with soft tissue depression or signs of aging who underwent combined nanofat, PRF, and autologous fat structural transplantation to control patients (77 cases) who underwent traditional autologous fat transplantation. Facial soft tissue depression symptoms and skin texture were improved to a greater extent after nanofat transplants than after traditional transplants, and the nanofat group had an overall satisfaction rate above 90%. These data suggest that NFSCs function similarly to mesenchymal stem cells and share many of the biological characteristics of traditional fat stem cell cultures. Transplants that combine newly-isolated nanofat, which has a rich stromal vascular fraction (SVF), with PRF and autologous structural fat granules may therefore be a safe, highly-effective, and long-lasting method for remodeling facial contours and rejuvenating the skin.

CD54+ rabbit adipose-derived stem cells overexpressing HIF-1α facilitate vascularized fat flap regeneration.

Fat flap transplantation is frequently performed in patients suffering from soft tissue defects resulting from disease or trauma. This study explored the feasibility of constructing vascularized fat flaps using rabbit adipose-derived stem cells (rASCs) and collagen scaffolds in a rabbit model. We evaluated rASCs proliferation, paracrine function, adipogenesis, vascularization, and CD54 expression, with or without HIF-1α transfection in vitro and in vivo. We observed that adipogenic differentiation potential was greater in rASCs with high CD54 expression (CD54+rASCs) than in those with low expression (CD54-rASCs), both in vitro and in vivo. HIF-1α overexpression not only augmented this effect, but also enhanced cell proliferation and paracrine function in vitro. We also demonstrated that HIF-1α-transfected CD54+rASCs showed enhanced paracrine function and adipogenic capacity, and that paracrine function increases expression of angiogenesis-related markers. Thus, CD54+rASCs overexpressing HIF-1α enhanced large volume vascularized fat flap regeneration in rabbits, suggesting CD54 may be an ideal candidate marker for ASCs adipogenic differentiation.

Ginsenoside Rg1 and platelet-rich fibrin enhance human breast adipose-derived stem cell function for soft tissue regeneration.

Adipose-derived stem cells (ASCs) can be used to repair soft tissue defects, wounds, burns, and scars and to regenerate various damaged tissues. The cell differentiation capacity of ASCs is crucial for engineered adipose tissue regeneration in reconstructive and plastic surgery. We previously reported that ginsenoside Rg1 (G-Rg1 or Rg1) promotes proliferation and differentiation of ASCs in vitro and in vivio. Here we show that both G-Rg1 and platelet-rich fibrin (PRF) improve the proliferation, differentiation, and soft tissue regeneration capacity of human breast adipose-derived stem cells (HBASCs) on collagen type I sponge scaffolds in vitro and in vivo. Three months after transplantation, tissue wet weight, adipocyte number, intracellular lipid, microvessel density, and gene and protein expression of VEGF, HIF-1α, and PPARγ were higher in both G-Rg1- and PRF-treated HBASCs than in control grafts. More extensive new adipose tissue formation was evident after treatment with G-Rg1 or PRF. In summary, G-Rg1 and/or PRF co-administration improves the function of HBASCs for soft tissue regeneration engineering.

Effect of activated autologous platelet-rich plasma on proliferation and osteogenic differentiation of human adipose-derived stem cells in vitro.

To investigate whether activated autologous platelet-rich plasma (PRP) can promote proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs) in vitro. hASCs were isolated from lipo-aspirates, and characterized by specific cell markers and multilineage differentiation capacity after culturing to the 3(rd) passage. PRP was collected and activated from human peripheral blood of the same patient. Cultured hASCs were treated with normal osteogenic inductive media alone (group A, control) or osteogenic inductive media plus 5%, 10%, 20%, 40%PRP (group B, C, D, E, respectively). Cell proliferation was assessed by CCK-8 assay. mRNA expression of osteogenic marker genes including alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN) and core binding factor alpha 1 (Cbfa1) were determined by Real-Time Quantitative PCR Analysis (qPCR). Data revealed that different concentrations of activated autologous PRP significantly promoted hASCs growth in the proliferation phase compared to the without PRP group and resulted in a dose-response relationship. At 7-d and 14-d time point of the osteogenic induced stage, ALP activity in PRP groups gradually increased with the increasing of concentrations of PRP and showed that dose-response relationship. At 21-d time point of the osteogenic induced stage, PRP groups make much more mineralization and mRNA relative expression of ALP, OPN, OCN and Cbfa1 than that without PRP groups and show that dose-response relationship. This study indicated that different concentrations of activated autologous PRP can promote cell proliferation at earlier stage and promote osteogenic differentiation at later stage of hASCs in vitro. Moreover, it displayed a dose-dependent effect of activated autologous PRP on cell proliferation and osteogenic differentiation of hASCs in vitro.

Human breast adipose­derived stem cells: characterization and differentiation into mammary gland­like epithelial cells promoted by autologous activated platelet­rich plasma.

Human adipose­derived stem cells (ASCs) isolated from various body sites have been widely investigated in basic and clinical studies. However, ASCs derived from human breast tissue (hbASCs) have not been extensively investigated. In order to expand our understanding of hbASCs and examine their potential applications in stem cell research and cell­based therapy, hbASCs were isolated from discarded surgical fat tissue following reduction mammoplasty and a comprehensive characterization of these hbASCs was performed, including analysis of their cellular morphology, growth features, cell surface protein markers and multilineage differentiation capacity. These hbASCs expressed cluster of differentiation (CD)44, CD49d, CD90 and CD105, but did not express CD31 and CD34. Subsequently, the hbASCs were differentiated into adipocytes, osteocytes and chondrocytes in vitro. In order to examine the potential applications of hbASCs in breast reconstruction, an approach to promote in vitro differentiation of hbASCs into mammary gland­like epithelial cells (MGECs) was developed using activated autologous platelet­rich plasma (PRP). A proliferation phase and a subsequent morphological conversion phase were observed during this differentiation process. PRP significantly promoted the growth of hbASCs in the proliferation phase and increased the eventual conversion rate of hbASCs into MGECs. Thus, to the best of our knowledge, the present study provided the first comprehensive characterization of hbASCs and validated their multipotency. Furthermore, it was revealed that activated autologous PRP was able to enhance the differentiation efficiency of hbASCs into MGECs. The present study and other studies of hbASCs may aid the development of improved breast reconstruction strategies.