Shaohua Chang

Paclitaxel impairs adipose stem cell proliferation and differentiation

BACKGROUND Cancer patients with chemotherapy-induced immunosuppression have poor surgical site wound healing. Prior literature supports the use of human adipose-derived stem cell (hASC) lipoinjection to improve wound healing. It has been established that multipotent hASCs facilitate neovascularization, accelerate epithelialization, and quicken wound closure in animal models. Although hASC wound therapy may benefit surgical cancer patients, the chemotherapeutic effects on hASCs are unknown. We hypothesized that paclitaxel, a chemotherapeutic agent, impairs hASC growth, multipotency, and induces apoptosis. METHODS hASCs were isolated and harvested from consented, chemotherapy and radiation naive patients. Growth curves, MTT (3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide), and EdU (5-ethynyl-2-deoxyguridine) assays measured cytotoxicity and proliferation. Oil Red O stain, Alizarin Red stain, matrigel tube formation assay, and quantitative polymerase chain reaction analyzed hASC differentiation. Annexin V assay measured apoptosis. Immunostaining and Western blot determined tumor necrosis factor α (TNF-α) expression. RESULTS hASCs were selectively more sensitive to paclitaxel (0.01-30 μM) than fibroblasts (P < 0.05). After 12 d, paclitaxel caused hASC growth arrest, whereas control hASCs proliferated (P = 0.006). Paclitaxel caused an 80.6% reduction in new DNA synthesis (P < 0.001). Paclitaxel severely inhibited endothelial differentiation and capillary-like tube formation. Differentiation markers, lipoprotein lipase (adipogenic), alkaline phosphatase (osteogenic), CD31, and van Willebrand factor (endothelial), were significantly decreased (all P < 0.05) confirming paclitaxel impaired differentiation. Paclitaxel was also found to induce apoptosis and TNF-α was upregulated in paclitaxel-treated hASCs (P < 0.001). CONCLUSIONS Paclitaxel is more cytotoxic to hASCs than fibroblasts. Paclitaxel inhibits hASC proliferation, differentiation, and induces apoptosis, possibly through the TNF-α pathway. Paclitaxels severe inhibition of endothelial differentiation indicates neovascularization disruption, possibly causing poor wound healing in cancer patients receiving chemotherapy.

Sphingosine-1-phosphate promotes the differentiation of adipose-derived stem cells into endothelial nitric oxide synthase (eNOS) expressing endothelial-like cells.

BackgroundAdipose tissue provides a readily available source of autologous stem cells. Adipose-derived stem cells (ASCs) have been proposed as a source for endothelial cell substitutes for lining the luminal surface of tissue engineered bypass grafts. Endothelial nitric oxide synthase (eNOS) is a key protein in endothelial cell function. Currently, endothelial differentiation from ASCs is limited by poor eNOS expression. The goal of this study was to investigate the role of three molecules, sphingosine-1-phosphate (S1P), bradykinin, and prostaglandin-E1 (PGE1) in ASC endothelial differentiation. Endothelial differentiation markers (CD31, vWF and eNOS) were used to evaluate the level of ASCs differentiation capability.ResultsASCs demonstrated differentiation capability toward to adipose, osteocyte and endothelial like cell phenotypes. Bradykinin, S1P and PGE were used to promote differentiation of ASCs to an endothelial phenotype. Real-time PCR showed that all three molecules induced significantly greater expression of endothelial differentiation markers CD31, vWF and eNOS than untreated cells. Among the three molecules, S1P showed the highest up-regulation on endothelial differentiation markers. Immunostaining confirmed presence of more eNOS in cells treated with S1P than the other groups. Cell growth measurements by MTT assay, cell counting and EdU DNA incorporation suggest that S1P promotes cell growth during ASCs endothelial differentiation. The S1P1 receptor was expressed in ASC-differentiated endothelial cells and S1P induced up-regulation of PI3K.ConclusionsS1P up-regulates endothelial cell markers including eNOS in ASCs differentiated to endothelial like cells. This up-regulation appears to be mediated by the up-regulation of PI3K via S1P1 receptor. ASCs treated with S1P offer promising use as endothelial cell substitutes for tissue engineered vascular grafts and vascular networks.

Fibroblast growth factor and vascular endothelial growth factor play a critical role in endotheliogenesis from human adipose-derived stem cells

Objective: Adipose‐derived stem cells (ASCs) are a potential adult mesenchymal stem cell source for restoring endothelial function in patients with critical limb ischemia. Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF) play a major role in angiogenesis and wound healing. This study evaluated the effects of FGF and VEGF on the proliferation, migration, and potential endothelial differentiation of human ASCs with regards to their use as endothelial cell substitutes. Methods: ASCs were isolated from clinical lipoaspirates and cultured in M199 medium with fetal bovine serum (10%), FGF2 (10 ng/mL), VEGF (50 ng/mL), or combinations of FGF2 and VEGF. Cell proliferation rates, viability, and migration were measured by growth curves, MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide), and scratch assays. For cell attachment determinations, ASCs were seeded onto a scaffold of small intestinal submucosa for 5 days. Endothelial differentiation capabilities of ASCs were confirmed by expression of endothelial cell‐specific markers using quantitative polymerase chain reaction, immunofluorescence staining, and cord formation on Matrigel (BD Biosciences, San Jose, Calif). PD173074, a selective inhibitor of FGF receptor, was used to confirm the importance of FGF signaling. Results: ASCs treated with FGF or combinations of FGF and VEGF showed increased proliferation rates and consistent differentiation toward an endothelial cell lineage increase in platelet endothelial cell adhesion molecule (CD31), von Willebrand factor, endothelial nitric oxide synthase, and vascular endothelial cadherin message, and in protein and cord formation on Matrigel. FGF and VEGF stimulated ASC migration and increased the attachment and retention after seeding onto a matrix graft of small intestinal submucosa. Blockade of FGF signaling with PD173074 abrogated ASC endothelial cell differentiation potential. Conclusions: These results indicate that FGF and VEGF are ASC promoters for proliferation, migration, attachment, and endothelial differentiation. FGF and VEGF have a costimulatory effect on ASC endotheliogenesis. These results further suggest that ASCs with enhanced FGF signaling may potentially be used for tissue engineering and cell‐based therapies in patients with critical limb ischemia. Clinical Relevance: Endothelial dysfunction is a primary mediator of vascular disease and critical limb ischemia. Treatment of this disease state is limited by available conduit and the inherent thrombogenicity of bypass grafts. Development of phenotypically and functionally similar endothelial cells from adipose‐derived stem cells may allow for the creation of vascular conduits that are readily available and provide increased patency rates. Our study differentiated a cell line from human adipose that could be manipulated into endothelial cells that exhibit characteristics of endothelial cells, providing further information to already existing literature.

Evaluation of function and recovery of adipose-derived stem cells after exposure to paclitaxel

BACKGROUND AIMS Adipose-derived stem cells (ASCs) are considered to play a positive role in wound healing as evidenced by their increasing use in breast reconstructive procedures. After chemotherapy for breast cancer, poor soft tissue wound healing is a major problem. In the present study, the functional capabilities and recovery of ASCs after exposure to chemotherapeutic agent paclitaxel (PTX) using in vitro and ex vivo models were demonstrated. METHODS Human ASCs were isolated from periumbilical fat tissue and treated with PTX at various concentrations. Adult Sprague-Dawley rats were given intravenous injections with PTX. Two and four weeks after the initial PTX treatment, ASCs were isolated from rat adipose tissue. Proliferation, cell viability, apoptosis and cell migration rates were measured by growth curves, MTT assays, flow cytometry and scratch assays. ASCs were cultured in derivative-specific differentiation media with or without PTX for 3 weeks. Adipogenic, osteogenic and endothelial differentiation levels were measured by quantitative reverse transcriptase polymerase chain reaction and histological staining. RESULTS PTX induced apoptosis, decreased the proliferation and cell migration rates of ASCs and inhibited ASCs multipotent differentiation in both in vitro human ASC populations and ex vivo rat ASC populations with PTX treatment. Furthermore, after cessation of PTX, ASCs exhibited recovery potential of differentiation capacity in both in vitro and animal studies. CONCLUSIONS Our results provide insight into poor soft tissue wound healing and promote further understanding of the potential capability of ASCs to serve as a cell source for fat grafting and reconstruction in cancer patients undergoing chemotherapy treatment.

In vitro effects of tamoxifen on adipose‐derived stem cells

In breast reconstructive procedures, adipose‐derived stem cells (ASCs) that are present in clinical fat grafting isolates are considered to play the main role in improving wound healing. In patients following chemotherapy for breast cancer, poor soft tissue wound healing is a major problem. However, it is unclear if tamoxifen (TAM) as the most widely used hormonal therapeutic agent for breast cancer treatment, affects the ASCs and ultimately wound healing. This study evaluated whether TAM exposure to in vitro human ASCs modulate cellular functions. Human ASCs were isolated and treated with TAM at various concentrations. The effects of TAM on cell cycle, cell viability and proliferation rates of ASCs were examined by growth curves, MTT assay and BrdU incorporation, respectively. Annexin V and JC‐1 Mitochondrial Membrane Potential assays were used to analyze ASC apoptosis rates. ASCs were cultured in derivative‐specific differentiation media with or without TAM (5 uM) for 3 weeks. Adipogenic and osteogenic differentiation levels were measured by quantitative RT‐PCR and histological staining. TAM has cytotoxic effects on human ASCs through apoptosis and inhibition of proliferation in dose‐ and time‐dependent manners. TAM treatment significantly down‐regulates the capacity of ASCs for adipogenic and osteogenic differentiation (p<0.05 vs. control), and inhibit the ability of the ASCs to subsequently formed cords in Matrigel. This study is the first findings to our knowledge that demonstrated that TAM inhibited ASC proliferation and multi‐lineage ASC differentiation rates. These results may provide insight into the role of TAM with associated poor soft tissue wound healing and decreased fat graft survival in cancer patients receiving TAM.

Elevated autophagy and mitochondrial dysfunction in the Smith–Lemli–Opitz Syndrome

Smith–Lemli–Opitz Syndrome (SLOS) is a congenital, autosomal recessive metabolic and developmental disorder caused by mutations in the enzyme which catalyzes the reduction of 7-dehydrocholesterol (7DHC) to cholesterol. Herein we show that dermal fibroblasts obtained from SLOS children display increased basal levels of LC3B-II, the hallmark protein signifying increased autophagy. The elevated LC3B-II is accompanied by increased beclin-1 and cellular autophagosome content. We also show that the LC3B-II concentration in SLOS cells is directly proportional to the cellular concentration of 7DHC, suggesting that the increased autophagy is caused by 7DHC accumulation secondary to defective DHCR7. Further, the increased basal LC3B-II levels were decreased significantly by pretreating the cells with antioxidants implicating a role for oxidative stress in elevating autophagy in SLOS cells. Considering the possible source of oxidative stress, we examined mitochondrial function in the SLOS cells using JC-1 assay and found significant mitochondrial dysfunction compared to mitochondria in control cells. In addition, the levels of PINK1 which targets dysfunctional mitochondria for removal by the autophagic pathway are elevated in SLOS cells, consistent with mitochondrial dysfunction as a stimulant of mitophagy in SLOS. This suggests that the increase in autophagic activity may be protective, i.e., to remove dysfunctional mitochondria. Taken together, these studies are consistent with a role for mitochondrial dysfunction leading to increased autophagy in SLOS pathophysiology.

The endothelial cell secretome as a novel treatment to prime adipose-derived stem cells for improved wound healing in diabetes

Background: Chronic wounds are a common surgical problem exacerbated by diabetes and ischemia. Although adipose‐derived stem cells (ASCs) have shown promise as a wound healing therapy, their function and proliferation are hindered in diabetes. This study examines the ability of the human umbilical vein endothelial cell (HUVEC) secretome to reverse the deleterious effects of high glucose concentrations on ASCs through priming, thereby enhancing their ability to participate in angiogenesis and wound healing. Methods: Institutional review board‐approved human ASCs were cultured in M199 medium with or without glucose (30 mmol/L). HUVEC were grown in 30 mmol/L glucose‐containing M199 medium; the resulting conditioned medium (HUVEC‐CM) was collected every 3 days and used to prime ASCs. An aliquot of HUVEC‐CM was heated (85°C for 30 minutes) to produce thermal denaturation of protein. Viability, proliferation, and endothelial differentiation were measured by MTT assays, growth curves, and quantitative polymerase chain reaction, respectively. A Matrigel assay was used to assess the ability of primed ASCs to participate in capillary‐like tube formation. An Institutional Animal Care and Use Committee–approved in vivo murine model of diabetic and ischemic hindlimbs was used to evaluate the angiogenic potential of primed stem cells. Human ASCs were cultured with either control M199 or HUVEC‐CM. Mice were randomized to a control group, an unprimed ASC group, or a HUVEC‐primed ASC group. Cellular therapies were injected into the ischemic muscle. Thirty days later, slides were made. Microvessels were counted by three blinded observers. Results: MTT assays revealed that HUVEC‐priming induced a 1.5 times increase in cell viability over diabetic controls. This promoting effect was lost with heated HUVEC‐CM (P < .001), indicating that the active molecules are of protein origin. After 9 days, ASCs cultured in 30 mmol/L glucose solution showed a 14% reduction in growth from nondiabetic controls (P = .013) and exhibited atrophic morphology. Conversely, diabetic HUVEC‐primed stem cells demonstrated a nearly four‐fold increase in proliferation (P < .05) and took on a fusiform, endothelial‐like phenotype. Polymerase chain reaction demonstrated enhanced expression of CD31 messenger RNA by 4.7‐fold after 14 days in the HUVEC‐primed group, and endothelial nitric oxide synthase messenger RNA messenger RNA was increased 20.1‐fold from controls. Unlike unprimed controls, HUVEC‐primed ASCs readily formed capillary‐like tube networks on Matrigel. Diabetic mice that were injected with HUVEC‐primed ASCs demonstrated greater vessel density than both controls (2.1‐fold) and unprimed stem cell treatments (P < .001). Conclusions: HUVECs secrete protein factors that significantly increase proliferation and endothelial differentiation of ASCs under diabetic conditions. Injection of ischemic hindlimbs in diabetic mice with HUVEC‐primed ASCs leads to enhanced angiogenesis. Clinical Relevance: In the current climate of increasing diabetes rates, there exists a significant need for improved therapies for diabetic wounds. ASCs have exhibited improved wound healing in previous studies, but this benefit is known to be suppressed by hyperglycemia. Treating stem cells with the secreted protein cocktail of endothelial cells—secretomal priming—seems to overcome the inhibition of the diabetic state, making ASCs a viable treatment option for these wounds. Priming evidently induces endothelial differentiation of ASCs, thereby enhancing their involvement in angiogenesis. This study demonstrates the potential of these primed ASC‐based therapies in vitro and in vivo and proposes several approaches by which they could be used in the clinical setting.

Endothelial Differentiated Adipose-Derived Stem Cells Improvement of Survival and Neovascularization in Fat Transplantation

Background Adipose-derived stem cells (ASCs) assisted lipotransfer have been considered to facilitate the survival of fat grafts. However, emerging evidence of insufficient vascularization is another obstacle for fat graft survival in cell-assisted lipotransfer. Objectives This study evaluated if endothelial phenotype ASCs with fat lipoaspirate improves survival and neovascularization in fat transplantation. Methods ASCs were isolated from human periumbilical fat tissue and cultured in endothelial growth medium for 2 weeks. Fat lipoaspirate was mixed with fresh adipose stroma vascular fraction (SVF), endothelial differentiated ASCs (EC/ASCs), and fat lipoaspirate alone. Three fat mixtures were subcutaneously injected into the adult male Sprague-Dawley rats dorsum at 3 locations. At 8 weeks after transplantation, the grafted fat lipoaspirates were harvested, and the extracted fat was evaluated using photographic, survival weights measurements and histological examination. Neo-vascularization was quantified by immunofluorescence and real-time RT-PCR. Results Grafts from the EC/ASC assisted group had a higher survival rate, morphologic integrity, and most uniform lipid droplets. They also revealed less inflammation and fibrosis with increased number of vessels by histological and immunofluorescence analysis. Quantitative RT-PCR analysis indicated that the expression levels of EC-specific markers of CD31 and vWF were higher in the EC/ASC group compared with in the control and fat with SVF transplants. Conclusions These results indicated that co-implantation of fat lipoaspirate with ASCs differentiated toward an endothelial phenotype improves both survival and neovascularization of the transplanted fat lipoaspirate, which might provide benefits and represents a promising strategy for clinical application in autologous fat transplantation.

Abstract 4077: Adipose-derived stem cells after Paclitaxel treatment demonstrate decreased function and suppression of breast cancer cell viability

Background: Following neoadjuvant or adjuvant chemotherapy for breast cancer resections, poor soft tissue wound healing can pose a major clinical challenge. Adipose-derived stem cells (ASCs) in clinically applied stromal vascular fractions are considered to play a positive role in wound healing (SVF) as evidenced by their increasing use in breast reconstructive procedures. Our study evaluated the potential confounding effects of Paclitaxel (PTX) on ASC proliferation and differentiation capacities and if PTX treated ASCs may subsequently influence tumor cell viability. Methods: IRB-approved human ASCs were isolated and treated with PTX at different concentrations. Proliferation, cell viability, and cell migration rates were measured by growth curves, MTT assays and scratch assays. ASCs were cultured in derivative-specific differentiation media with or without PTX (1uM) for 3 weeks. Adipogenic, osteogenic and endothelial differentiation levels were measured by quantitative RT-PCR, Oil-Red-O, Alizarin Red staining and cord formation on Matrigel, respectively. MDA-MB-231 (breast cancer cell line) and ASCs were co-cultured at equal cell numbers. ASC conditional media were collected every 3 days from ASC no-PTX treatment and PTX treated - washout groups. Results: PTX decreased proliferation of ASCs in a dose- and time-dependent manner. PTX treatment down-regulated the capacity of ASCs for adipogenic, osteogenic and endothelial differentiation (p Conclusion: Our results indicate that: 1) PTX inhibited ASC proliferation and decreased the multipotency differentiating capacity; and 2) Direct contact or treatment with conditional media from PTX treated ASCs suppressed tumor cell growth. In evaluating autologous ASCs for reconstructive procedures, this study may provide insight into poor soft tissue wound healing immediately following the removal of chemotherapy as well as decreased recurrence in breast cancer patients. Citation Format: William M. Harris, Michael Plastini, Telisha Ortiz, Nikolas Kappy, Jefferson Benites, Shaohua Chang, A. Lelani Fahey, Martha S. Matthews, Alexandre Hageboutros, Jeffrey P. Carpenter, Spencer Brown, Ping Zhang. Adipose-derived stem cells after Paclitaxel treatment demonstrate decreased function and suppression of breast cancer cell viability. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4077. doi:10.1158/1538-7445.AM2015-4077