Non-contact Co-culture System Research Articles

Establishment of invitro model of erectile dysfunction for the study of high-glucose-induced angiopathy and neuropathy.

Penile erection requires complex interaction between vascular endothelial cells, smooth muscle cells, pericytes, and autonomic nerves. Diabetes mellitus is one of the most common causes of erectile dysfunction (ED) and multiple pathogenic factors, such as cavernous angiopathy and autonomic neuropathy, are associated with diabetic ED. Although a variety of animal models of diabetic ED play an important role in understanding pathophysiologic mechanisms of diabetes-induced ED, these animal models have limitations for addressing the exact cellular or molecular mechanisms involved in ED. Therefore, we established an invitro model of ED for the study of high-glucose-induced angiopathy and neuropathy. We successfully isolated and cultivated mouse cavernous endothelial cells (MCECs) and mouse cavernous pericytes (MCPs). The cells were exposed to the normal-glucose (5mmoL) or high-glucose (30mmoL) condition for 48h. In vitro matrigel assay revealed impairments in tube formation in primary cultured MCECs or MCPs exposed to high-glucose condition. To study cellular interaction between MCECs and MCPs, co-culture systems including indirect contact, indirect non-contact, and direct mixed co-culture system, were established. We observed impaired tube formation and increased permeability in MCECs-MCPs co-culture exposed to high-glucose condition. To evaluate the effect of high-glucose on neurite sprouting, the mouse major pelvic ganglion (MPG) tissue was harvested and cultivated in matrigel. Neurite outgrowth and nNOS-positive nerve fibers were significantly lower in MPG tissues exposed to the high-glucose condition than in the tissues exposed to the normal-glucose condition. We believe that invitro model of ED will aid us to understand the role of each cellular component in the pathogenesis of diabetic ED, and also be a useful tool for determining the efficacy of candidate therapeutics targeting vascular or neuronal function. This model would present a new avenue for drug discovery and development of novel therapeutic modalities for erectile dysfunction.

Influences of human dermis derived mesenchymal stem cells on α-SMA and DCN expressions of hypertrophic scars fibroblasts

To preliminarily explore the effects of human dermis derived mesenchymal stem cells (hDMSCs) on expressions of α-smooth muscle actin (α-SMA) and decorin (DCN) in hypertrophic scars fibroblasts (HSFB) at different periods,and to explore the feasibility of MSCs in prevention and treatment of HSFBs. hDMSCs were cultured with mechanical method combined with enzyme digestion.The cells of the third generation which were well grown were taken,and flow cytometry (FCM) was used to detect CD molecules in hDMSCs.Immunocytochemistry was used to detect cytokeratin 19 (CK19) and vimentin and identify the separated cells.The cells were differentiated into lipoblasts,chondroblasts and osteoblasts.According to the formation course of hypertrophic scar,the scar specimens were divided into 6-month,l-year,and 2-year group with three cases in each group.HSFBs from different groups were co-cultured with well-adherent hDMSCs of the third generation in non-contact transwell co-culture system for 21 days. And HSFBs from the corresponding groups were cultured in normal six-well plate as the controls.Real-time fluorescent-polymerase chain reaction (RT-PCR) and Western Blot were used to detect the expressions of mRNA and proteins of α-SMA and DCN in HSFBs from different groups. hDMSCs highly expressed the surface markers including CD73,CD105,CD44 and CD90,etc.,but did not express hematopoietic stem cell surface markers including CD 14,CD34 and CD45.They positively expressed vimentin but not CK19.The cells can be differentiated into lipoblasts,chondroblasts and osteoblasts,which was in line with the minimum identification standards of mesenchymal stem cells.For HSFB cultured in normal six-well plates,α-SMA mRNA and protein expressions of HSFB in the 6-month,1-year and 2-year groups were 198.20 ± 15.46/0.29 ± 0.070,175.24 ± 17.04/0.38 ± 0.110,and 125.73 ± 6.99/0.33 ±0.085,respectively;while DCN mRNA and protein expressions of HSFB in the corresponding groups were 61.30 ± 9.79/0.015 ± 0.003,70.89 ± 11.29/0.020 ± 0.007,and 77.31 ± 4.80/0.023 ± 0.003,respectively.For HSFB co-cultured with 5 × 104 hDMSCs, oα-SMA mRNA and protein expressions of HSFB in the 6-month,1-year and 2-year groups were 48.40 ± 6.42/ 0.100 ± 0.020,192.16 ± 11.37/0.110 ± 0.014,and 73.33 ± 6.29/0.110 ± 0.016,respectively;while DCN mRNA and protein expressions of HSFB in the corresponding groups were 156.92 ± 14.91/0.049 ±0.015,154.42 ± 18.17/0.033 ± 0.008,and 140.82 ± 7.32/0.030 ± 0.004,respectively.Compared with the control group(Cultured in normal six-well plates), mRNA and protein expressions of α-SMA in HSFBs were decreased after co-culture with 5 × 104 hDMSCs, mRNA and protein expressions of DCN were increased. Furthermore, it suggested that hypertrophic scar changed significantly in the early formation stage namely in the 6-month group. hDMSCs can down-regulate α-SMA mRNA and protein expressions of HSFBs and up-regulate mRNA and protein expressions of DCN in the in-vitro culture system.Those effects were particularly obvious on fibroblasts at the early formation of hyperplastic scar.Anti fibrosis role of hDMSCs is expected to be used in increasing the healing quality of the wound and in the prevention and treatment of pathological scars.

Triptolide represses oral cancer cell proliferation, invasion, migration, and angiogenesis in co-inoculation with U937 cells

ObjectivesAdvanced oral cancer is a major public health concern because of a lack of effective prevention and treatment. Triptolide (TPL), a diterpenoid triepoxide derived from the Chinese herb Tripterygium wilfordii, has been demonstrated to possess strong anticancer properties. In this study, we investigated whether TPL exerts anticancer effects on the tumor microenvironment of head and neck squamous cell carcinoma (HNSCC).Materials and methodsHuman macrophage-like U937 cells were co-inoculated with oral cancer SAS cells in a noncontact transwell coculture system. Cytokine expression was detected using ELISA, and cell proliferation was detected using methylene blue. RNA levels were detected using qPCR. Protein levels were detected using Western blot analysis. In vivo experiments involved using xenografted NOD/SCID mice.ResultsOur results demonstrated that TPL inhibited the growth of SAS cells co-inoculated with U937 cells in vitro and in vivo. TPL inhibited the invasion, migration ability, and angiogenesis of SAS cells co-inoculated with U937 cells. Expression of cytokines IL-6, IL-8, and TNF-α was induced by co-inoculation, but TPL repressed their expression.ConclusionTPL suppressed the expression of cytokines IL-6, IL-8, and TNF-α, as well as tumor growth, invasion, migration, and angiogenesis in the co-inoculation of human tongue cancer cells with macrophage-like U937 cells.Clinical relevanceTPL is a potential candidate among novel chemotherapeutic agents or adjuvants for modulating tumor-associated macrophages in a tumor microenvironment of HNSCC.

Hydroxysafflor Yellow A Attenuates Neuron Damage by Suppressing the Lipopolysaccharide-Induced TLR4 Pathway in Activated Microglial Cells.

Microglia activation initiates a neurological deficit cascade that contributes to substantial neuronal damage and impairment following ischemia stroke. Toll-like receptor 4 (TLR4) has been demonstrated to play a critical role in this cascade. In the current study, we tested the hypothesis that hydroxysafflor yellow A (HSYA), an active ingredient extracted from Flos Carthami tinctorii, alleviated inflammatory damage, and mediated neurotrophic effects in neurons by inducing the TLR4 pathway in microglia. A non-contact Transwell co-culture system comprised microglia and neurons was treated with HSYA followed by a 1mg/mL lipopolysaccharide (LPS) stimulation. The microglia were activated prior to neuronal apoptosis, which were induced by increasing TLR4 expression in the activated microglia. However, HSYA suppressed TLR4 expression in the activated microglia, resulting in less neuronal damage at the early stage of LPS stimulation. Western blot analysis and immunofluorescence indicated that dose-dependently HSYA down-regulated TLR4-induced downstream effectors myeloid differentiation factor 88 (MyD88), nuclear factor kappa b (NF-κB), and the mitogen-activated protein kinases (MAPK)-regulated proteins c-Jun NH2-terminal protein kinase (JNK), protein kinase (ERK) 1/2 (ERK1/2), p38 MAPK (p38), as well as the LPS-induced inflammatory cytokine release. However, HSYA up-regulated brain-derived neurotrophic factor (BDNF) expression. Our data suggest that HSYA could exert neurotrophic and anti-inflammatory functions in response to LPS stimulation by inhibiting TLR4 pathway-mediated signaling.

Effect of human umbilical cord mesenchymal stem cells on endometriotic cell proliferation and apoptosis.

The objective of this study was to observe the effects of human umbilical cord mesenchymal stem cells (UCMSCs) on the proliferation and apoptosis of endometriotic cells. Endometriotic cells and UCMSCs were primarily cultured in vitro. In the experimental group, a UCMSC and endometriotic cell non-contact co-culture system was established. The control group consisted of 1 x 10(5) endometriotic cells cultured alone. The proliferation and apoptosis of endometriotic cells were respectively detected using the MTT method and flow cytometry. The mRNA expression level of the tensin homologue gene (PTEN) in endometriotic cells was detected by reverse transcription-polymerase chain reaction amplification. Compared with the control group, the proliferation of endometriotic cells in the experimental group was clearly inhibited (P < 0.05) and time-dependent (P < 0.05). In addition, the number of apoptotic cells were significantly increased (P < 0.05), and the amount of cells, which entered S phase from G1 phase, decreased significantly. Furthermore, the mRNA expression level of the PTEN gene in the experimental group was significantly higher than in the control group (P < 0.05). These results suggest that UCMSCs might inhibit the proliferation of human endometriotic cells in vitro and promote their apoptosis by upregulating the expression of PTEN.

Exosomes Derived from AML/MDS Cells Is Involved in Stromal Dysfunction and Bone Marrow Failure

The failure of normal hematopoiesis in myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) could be induced by a variety of mechanism such as the alteration of property of hematopoietic stem cells and stem cell niche. However, it has not yet been clarified precise mechanism how MDS stem/progenitor cells could replace normal hematopoietic stem/progenitor cells especially regarding involvement of mesenchymal stromal cells (MSCs). To gain insight into the mechanism of stromal dysfunction, comparative analyses of transcriptomes were conducted between normal and MDS/AML-derived MSCs. Further, we attempted to identify certain effectors originated from MDS/AML cells could alter the function of bone marrow (BM) MSCs.The MSCs derived from healthy volunteer (HV)-derived (normal) and MDS/AML-derived stromal cells were established and analyzed mRNA expression by quantitive PCR (qPCR) array. Additionally, the supporting activity of MSCs for BM CD34+ progenitor/stem cells was examined using serum free coculture system. The interaction between MDS/AML cells and MSCs were evaluated by using Boyden Chamber and the changes of mRNA expression were analyzed.The results of qPCR array revealed that the expression of hematopoietic factors was drastically altered in MDS/AML-derived MSCs as compared with normal MSCs. Among these factors, the expression of SCF and JAG1 mRNA were significantly and consistently reduced in all MDS/AML patients examined. Functional assay of these MSCs demonstrated that the number of colony-forming units (CFU) mixed cells (MIXs) and cobblestone area-forming cells (CAFCs) derived from CD34+ cells was significantly reduced after coculture with MDS/AML-derived MSCs as compared with normal MSCs. Even non-contact culture using Boyden Chamber between leukemic cells and MSCs induced the reduction of SCF and JAG1 mRNA, indicating that certain inducers could be soluble factors. Interestingly, this effect of transcriptomes alteration was negated by nSMase2 inhibitor (GW4869). Exosome transfer assay using Boyden Chamber revealed that GFP and PKH26 in leukemic cells transmit onto MSCs in non-contact coculture system and this transfer of exosome was significantly inhibited by GW4869 or nSMase siRNA. The multiple type of microRNA in exosome derived from MDS/AML cells was transferred into MSCs, suggesting that exosome could contribute to the alteration of mRNA expression in stromal cells.Collectively, these results indicated that exosome derived from MDS/AML cells could be involved in the reduction of SCF/JAG mRNA and the stromal supporting activity of normal hematopoietic stem/progenitor cells. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1035: hMENA splicing program and TGF-β1-mediated EMT in pancreatic cancer

Abstract Background Epithelial to mesenchymal transition (EMT) is an early event in pancreatic cancer and has been involved in cancer invasiveness. An intense stromal reaction, peculiar to the pancreatic tumor microenvironment, includes cancer-associated fibroblasts(CAFs), abundant cells in the tumor stroma, recently linked to the induction of EMT. On the other hand, the EMT process requires a dynamic remodeling of the actin cytoskeleton, and the splicing program of hMENA, a regulator of actin, has been associated with the EMT process. We have described two alternatively expressed isoforms, hMENA11a and hMENAΔv6, with opposite functions in invasiveness in breast cancer (1). hMENA expression was detected in human pancreatic ductal adenocarcinoma samples (PDAC) (2), but no data are available on the alternative isoform expression in this neoplasia. The aim of this study is to investigate the role of hMENA splicing in TGF-β -mediated EMT in pancreatic cancer, the mechanisms involved in hMENA induction in PDAC and the role of CAFs in this process. Methods hMENA isoform expression was evaluated in PDAC tissues by immunohistochemistry using isoform-specific antibodies. Human PDAC cell lines, untreated or TGF-β treated, were characterized for the expression of hMENA isoforms and markers of EMT by qRT-PCR and WB analysis. The effects of both hMENAΔv6 knockdown or overexpression were also evaluated. Pancreatic cancer associated-fibroblasts were isolated from primary PDAC tissues. To study the role of fibroblast-cancer cell interactions on hMENA expression, a noncontact coculture system was used. Results IHC analysis of PDAC tissues revealed that epithelial hMENA11a is rarely expressed in primary pancreatic tumors that express a high level of hMENA and hMENAΔv6 isoforms. In a panel of pancreatic cancer cell lines, hMENA11a expression correlates with an epithelial phenotype, wherea hMENAΔv6 expression is associated with a mesechymal phenotype. TGF-β treatment specifically upregulated the invasive hMENAΔv6 isoform expression. Knockdown of endogenous hMENA/hMENAΔv6 isoform reduced cell invasiveness, reverted cells to an epithelial -“like” phenotype with an increased E-cadherin expression and impaired the TGF-β-mediated vimentin up-regulation. Conversely, overexpression of hMENAΔv6 increased the expression of the mesenchymal marker vimentin. Freshly explanted CAFs expressed the “mesenchymal” hMENAΔv6, and not hMENA11a and produced paracrine factors involved in the induction of hMENA isoforms in tumor cells. Conclusions These data provide new and critical insights into the role of hMENA splicing in TGF-β mediated EMT and identify the hMENA splicing program as a promising pathway for the development of new diagnostics and therapeutics in PDAC. (1) Di Modugno F. et al PNAS 2012 (2) Pino S. et al Clin Cancer Res 2008 Citation Format: Roberta Melchionna, Pierluigi Iapicca, Francesca Di Modugno, Paola Trono, Novella Gualtieri, Maria Grazia Diodoro, Sheila Spada, Giuliana Falasca, Gian Luca Grazi, Mina J Bissell, Paola Nisticò. hMENA splicing program and TGF-β1-mediated EMT in pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1035. doi:10.1158/1538-7445.AM2014-1035

Abstract 3383: JunB/AP-1 controls MM cell proliferation, survival and drug resistance in the bone marrow microenvironment

Abstract The activator protein-1 (AP-1) transcription factor is a dimeric complex that comprises members of the JUN, FOS, activating transcription factor (ATF) and musculoaponeurotic fibrosarcoma (MAF) protein families. Resultant heterodimers and homodimers determine the genes that are regulated by AP-1. Functionally, AP-1 has been implicated in a multitude of biological processes but also tumorigenesis, dependent on the cell type and the pathophysiologic context. In multiple myeloma (MM), the role of AP-1 is largely unknown. The present study focuses on the impact of JunB, a central member of the AP-1 family, in MM growth, survival and drug resistance. First, our data demonstrate rapid and strong induction of AP-1/JunB expression in MM cell lines and primary tumor cells upon co-culture with bone marrow stromal cells (BMSCs), i.e. isotypic primary BMSCs as well as BMSC lines KM-105 and HS-27A. Importantly, utilizing a non-contact co-culture system, we show that this effect is predominantly mediated by soluble factors secreted by BMSCs rather than direct MM-BMSC contact. Consistent with gene expression profiling data, JunB mRNA levels detected by quantitative real-time PCR are increased in MM cells when co-cultured with BMSCs. Specifically, we found that upregulation of JunB occurs predominantly at the translational level, since cycloheximide, but not actinomycin D or MG-132 significantly blocked BMSC- induced JunB expression. Pharmacologic inhibition was used next in order to identify upstream signaling pathways, which mediate BMSC- induced JunB upregulation in MM cells. Our data demonstrate that activation of MEK/MAPK or NF-kB, but not PI3K/Akt is required for induction of JunB expression and AP-1 transcriptional activity. To delineate the specific functional role of JunB in MM pathogenesis, we subsequently generated stable MM cell lines carrying the pLKO.1-puro-JunB shRNA vector or the pLKO.1-puro control vector, respectively. Our preliminary data show significantly decreased proliferation and resistance to chemotherapy in MM/ pLKO.1-puro-JunB shRNA but not MM/ pLKO.1-puro control cells when co-cultured with BMSCs. Consistently, 4-hydroxytamoxifen (4-HT) treatment of MM cells stably transduced with pBabe-puro-JunB-ER-IRES-GFP but not of MM cells transduced with pBabe-puro-IRES-GFP promotes tumor cell proliferation, survival and drug resistance, independent of BMSC co-culture. Ongoing studies aim to identify downstream effectors of JunB, using gene array profiling of MM/ pLKO.1-puro-JunB shRNA cells versus MM/ pLKO.1-puro control cells. Furthermore, we are conducting a survey of published and unpublished datasets to uncover the clinical and prognostic relevance of JunB expression in MM patient samples. In summary, our data demonstrate for the first time an important role of AP-1/JunB in MM tumorigenesis and strongly propose it as a novel therapeutic target in MM. Citation Format: Fengjuan Fan, Sonia Vallet, Martin Sattler, Giovanni Tonon, Muhammad Hasan Bashari, Latifa Bakiri, Hartmut Goldschmidt, Erwin F. Wagner, Dirk Jaeger, Klaus Podar. JunB/AP-1 controls MM cell proliferation, survival and drug resistance in the bone marrow microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3383. doi:10.1158/1538-7445.AM2014-3383

Periodontal cell implantation contributes to the regeneration of the periodontium in an indirect way.

Periodontitis is the most common human infectious disease. Regeneration of bone and soft tissue defects after periodontitis remains challenging, although the transplantation of periodontal ligament (PDL) cells seems a liable strategy. However, little is known about the function of PDL cells after transplantation. In the current study, a combination of in vitro coculture systems and in vivo immunohistochemistry (IHC) was used to investigate the role of PDL cells in the regenerative process. First, a coculture method was used, in which mesenchymal cells (representing the host tissue) were brought into direct contact with PDL cells (representing the transplanted cell population). It was found that PDL cells significantly increased mineralized matrix formation and osteocalcin expression, whereas control cells did not. Similar results were obtained when a noncontact coculture system was applied separating PDL and mesenchymal cells. In an in vivo rat model, regeneration of alveolar bone and ligament was seen after PDL cell transplantation. Implanted PDL cells were found clustered along the newly formed tissues. IHC showed enhanced osteopontin expression and gap junction staining in areas neighboring implanted PDL cells. In conclusion, PDL cells enhance periodontal regeneration through a trophic factor stimulating the osteogenic activity of the surrounding host cells.

Luteolin Inhibits Smooth Muscle Cell Migration and Proliferation by Attenuating the Production of Nox4, P-Akt and VEGF in Endothelial Cells

The goals of this study were to observe how luteolin protects endothelial cells (ECs) from injury stimulated by Angiotensin II (Ang II), investigate the role of vascular endothelial dysfunction in vascular smooth muscle cell proliferation and migration in vitro and investigate its primary mechanism of action. A non-contact coculture system was established using a transwell system; ECs were cultured in the lower wells, while the smooth muscle cells (SMCs) were cultured in the upper wells. Cell proliferation was assessed by the MTT assay. The number of SMCs that migrated through the membrane of transwell system were observed and counted. The expression levels of various proteins (VEGF, p-Akt, Nox4) expressed in ECs were determined by Western blotting. VEGF mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). The supernatants of ECs were measured by enzyme linked immunosorbent assay (ELISA) to assay VEGF concentration. Ang II-stimulated ECs significantly increased the proliferation and migration of SMCs, and these effects were inhibited by luteolin pretreatment. Luteolin suppressed the Ang II-induced upregulation of Nox4, p-AKT and VEGF expression in ECs. These results demonstrate that luteolin is capable of inhibiting endothelial dysfunction induced by Ang II by suppressing the upregulation of Nox4, p-Akt and VEGF, thereby restraining the proliferation and migration of SMCs induced by injured ECs.

Role of bone marrow mesenchymal stem cells in restoring the functions of degenerative nucleus pulposus cells

To explore the effects of bone mesenchymal stem cells (BMSCs) in restoring the functions of degenerative nucleus pulposus cells (dNPCs). The animal models of degenerative nucleus pulposus were established by means of acupuncture and aspiration. The BMSCs as well as the normal and degeneratived nucleus pulposus cells of SD rats were isolated and cultured. The BMSCs/alginate gel complex and dNPCs/alginate gel complex were used for indirect co-culture in vitro, which was set as experiment group. The NPCs and dNPCs cultured alone as positive and negative controls. The cell growth conditions were observed by light microscopy, and suitable cells were selected to combine alginate gel stents and cultured in transwell plate. Seven days later, nucleus pulposus cells of each group were recycled, and the mRNA expressions of Collagen2, SOX 9, and Aggrecan were detected by RT-PCR, and the Collagen1, 2, and Aggrecan were detected by Western blotting and immunofluorescence. After non-contact co-culture for 7 days, the mRNA levels of Collagen2, SOX 9, and Aggrecan increased apparently in BMSCs+dNPCs group, while it was significantly lower in dNPCs sample (all P<0.05). The content of Collagen2 and Aggrecan detected by Western blotting in BMSCs+dNPCs group got close to NPCs sample, but it was significantly higher than dNPCs samples (all P<0.05), while the content of Collagen1in BMSCs+dNPCs group got close to NPCs samples, but it was significantly lower than dNPCs sample (P<0.05). Immunofluorescence results showed that cytoplasm was dyed red and the color near the caryon became dark in BMSCs+dNPCs group and dNPCs group;however, the colored scope of cytoplasm and the dark colored scope surrounding the caryon in BMSCs+dNPCs group was obviously larger than dNPCs group. Under a 3D non-contact co-culture system, BMSCs can promote the expression of epimatrix of the dNPCs, which shows that BMSCs can restore the functions of dNPCs of intervertebral disc to certain extent.

Technique for derivation of stem cell lines from the inner cell mass of mouse blastocsyts using a novel human endometrial cell line and a non-contact co-culture system

Therapeutic use of stem cells may require the derivation and propagation of clinical grade stem cell lines using non-animal feeder layers. To this end, we test a novel human endometrial cell (HEC) line as a feeder layer and its ability to support the growth of stem cells isolated from the inner cell mass (ICM) of mouse blastocsyts.

Development of a xeno-free non-contact co- culture system for derivation and maintenance of embryonic stem cells using a novel human endometrial cell line

PurposeMouse embryonic fibroblast feeder layers (MEF) have conventionally been used to culture and maintain the pluripotency of embryonic stem cells (ESC). This study explores the potential of using a novel human endometrial cell line to develop a non-xeno, non-contact co-culture system for ESC propagation and derivation. Such xeno-free systems may prove essential for the establishment of clinical grade human ESC lines suitable for therapeutic application.MethodsA novel line of human endometrial cells were seeded in a 6-well dish. Filter inserts containing mouse ESCs were placed on these wells and passaged 2–3 times per week. Inner cell masses derived from mouse blastocysts were also cultured on transwells in the presence of the feeder layer. In both cases, staining for SSEA-1, SOX-2, OCT-4 and alkaline phosphatase were used to monitor the retention of stem cells.ResultsESC colonies retained their stem cell morphology and attributes for over 120 days in culture and 44 passages to date. Inner cell mass derived ESC cultures were maintained in a pluripotent state for 45 days, through 6 passages with retention of all stem cell characteristics. The stem cell colonies expressed stem cell specific markers SSEA-1, Sox 2, Oct-4 and alkaline phosphatase. Upon removal of the human feeder layer, there was a distinct change in cell morphology within the colonies and evidence of ESC differentiation.ConclusionsHuman feeder layers offer a simple path away from the use of MEF feeder cells or MEF conditioned medium for ESC culture. Furthermore, indirect co-culture using porous membranes to separate the two cell types can prevent contamination of stem cell preparations with feeder cells during passaging.

Blocking the Interaction between Apolipoprotein E and Aβ Reduces Intraneuronal Accumulation of Aβ and Inhibits Synaptic Degeneration

Accumulation of β-amyloid (Aβ) in the brain is a key event in Alzheimer disease pathogenesis. Apolipoprotein (Apo) E is a lipid carrier protein secreted by astrocytes, which shows inherent affinity for Aβ and has been implicated in the receptor-mediated Aβ uptake by neurons. To characterize ApoE involvement in the intraneuronal Aβ accumulation and to investigate whether blocking the ApoE/Aβ interaction could reduce intraneuronal Aβ buildup, we used a noncontact neuronal-astrocytic co-culture system, where synthetic Aβ peptides were added into the media without or with cotreatment with Aβ12-28P, which is a nontoxic peptide antagonist of ApoE/Aβ binding. Compared with neurons cultured alone, intraneuronal Aβ content was significantly increased in neurons co-cultured with wild-type but not with ApoE knockout (KO) astrocytes. Neurons co-cultured with astrocytes also showed impaired intraneuronal degradation of Aβ, increased level of intraneuronal Aβ oligomers, and marked down-regulation of several synaptic proteins. Aβ12-28P treatment significantly reduced intraneuronal Aβ accumulation, including Aβ oligomer level, and inhibited loss of synaptic proteins. Furthermore, we showed significantly reduced intraneuronal Aβ accumulation in APPSW/PS1dE9/ApoE KO mice compared with APPSW/PS1dE9/ApoE targeted replacement mice that expressed various human ApoE isoforms. Data from our co-culture and in vivo experiments indicate an essential role of ApoE in the mechanism of intraneuronal Aβ accumulation and provide evidence that ApoE/Aβ binding antagonists can effectively prevent this process.

Effects of vascular endothelial cells on osteogenic differentiation of noncontact co‐cultured periodontal ligament stem cells under hypoxia

During periodontitis or orthodontic tooth movement, the periodontal vasculature is severely impaired by chronic inflammation or excessive mechanical force. This leads to a hypoxic microenvironment of the periodontal cells and enhances the expression of various cytokines and growth factors that may regulate angiogenesis and alveolar bone remodeling. However, the role of hypoxia in regulating the communication between endothelial cells (ECs) and osteoblast progenitors during the remodeling and repair of periodontal tissue is still poorly defined. The aim of this study was to investigate the effects of vascular ECs on osteogenic differentiation, mineralization and the paracrine function of noncontact co-cultured periodontal ligament stem cells (PDLSCs) under hypoxia, and further reveal the involvement of MEK/ERK and p38 MAPK pathways in the process. First, PDLSCs were obtained and a noncontact co-culture system of PDLSCs and human umbilical vein endothelial cells was established. Second, the effects of different time-periods of hypoxia (2% O(2) ) on the osteogenic potential, mineralization and paracrine function of co-cultured PDLSCs were investigated. Third, ERK1/2 and p38 MAPK activities of PDLSCs under hypoxia were measured by western blotting. Finally, we employed specific MAPK inhibitors (PD98059 and SB20350) to investigate the involvement of ERK1/2 and p38 MAPK in PDLSC osteogenesis under hypoxia. We observed further increased osteogenic differentiation of co-cultured PDLSCs, manifested by markedly enhanced alkaline phosphatase (ALP) activity and prostaglandin E(2) (PGE(2)) levels, vascular endothelial growth factor (VEGF) release, runt-related transcription factor 2 (Runx2) and Sp7 transcriptional and protein levels and mineralized nodule formation, compared with PDLSCs cultured alone. ERK1/2 was phosphorylated in a rapid but transient manner, whereas p38 MAPK was activated in a slow and sustained way under hypoxia. Furthermore, hypoxia-stimulated transcription and expression of osteogenic regulators (hypoxia-inducible factor-1α, ALP, Runx2, Sp7, PGE(2) and VEGF) were also inhibited by PD98059 and SB203580 to different degrees. Further increased osteogenic differentiation and mineralization of co-cultured PDLSCs under hypoxia were regulated by MEK/ERK and p38 MAPK pathways. And the ECs-mediated paracrine of PGE(2) and VEGF may facilitate the unidirectional PDLSC-EC communication and promote PDLSCs osteogenesis.

Morphological Assessment of Neurite Outgrowth in Hippocampal Neuron‐Astrocyte Co‐Cultures

Neurite outgrowth is a fundamental event in brain development, as well as in regeneration of damaged neurons. Astrocytes play a major role in neuritogenesis, by expressing and releasing factors that facilitate neurite outgrowth, such as extracellular matrix proteins, and factors that can inhibit neuritogenesis, such as the chondroitin sulfate proteoglycan neurocan. In this unit we describe a noncontact co-culture system of hippocampal neurons and cortical (or hippocampal) astrocytes for measurement of neurite outgrowth. Hippocampal pyramidal neurons are plated on glass coverslips, which are inverted onto an astrocyte feeder layer, allowing exposure of neurons to astrocyte-derived factors without direct contact between these two cell types. After co-culture, neurons are stained and photographed, and processes are assessed morphologically using Metamorph software. This method allows exposing astrocytes to various agents before co-culture in order to assess how these exposures may influence the ability of astrocytes to foster neurite outgrowth.

Zeaxanthin inhibits PDGF‐BB‐induced migration in human dermal fibroblasts

Zeaxanthin is the dihydroxy carotenoid and is distributed in our daily foods. Various natural carotenoids, including zeaxanthin, have been shown to inhibit proliferation of several types of cancer cells, but available data on the effect of zeaxanthin on skin fibroblasts and melanoma cells are limited. Platelet-derived growth factor (PDGF) functions as a chemotactic factor for dermal fibroblasts and plays an important role in the progression of melanoma. In this study, we investigated the effects of zeaxanthin on the migration of skin fibroblasts induced by PDGF-BB and melanoma cells. We demonstrated that zeaxanthin inhibited PDGF-BB-induced skin fibroblast migration on collagen and gelatin by a modified Boyden chamber system. The electric cell-substrate impedance sensing (ECIS) method also showed similar inhibitory effects of zeaxanthin on the migration of fibroblasts. In functional studies, zeaxanthin decreased melanoma-induced fibroblast migration in a non-contact coculture system and also the migration stimulated by melanoma-derived conditioned medium. Further analysis showed that zeaxanthin attenuated PDGF-BB and melanoma-conditioned medium induced phosphorylation of PDGFR-beta and MAP kinase in a concentration-dependent manner in human skin fibroblasts. However, these effects did not result from direct interaction of zeaxanthin with PDGF-BB. Thus, our results provide the first evidence showing that zeaxanthin is an effective inhibitor of migration of stromal fibroblasts induced by PDGF-BB and melanoma cells and this effect may further support its antitumor potential.

Bone Marrow–Derived Mesenchymal Stem Cells Promote Angiogenic Processes in a Time- and Dose-Dependent MannerIn Vitro

Bone marrow-derived mesenchymal stem cells (MSCs) have received much attention as a potential treatment for myocardial infarction because of their potential to integrate into the host myocardium and repair the injured heart. The mode of action of stem cell-mediated cardiac repair is still somewhat unclear, including the potential role of MSCs in neovascularization. The objective of this study was to determine the in vitro effect of MSCs on angiogenesis-related endothelial cell (EC) behavior, including migration, monolayer permeability, and vessel formation and stabilization. In a noncontact coculture system, we found that MSCs increase EC proliferation and migration, promoting early events of angiogenesis, while also decreasing EC monolayer permeability. Further, in a time- and dose-dependent manner, MSCs in direct coculture with ECs on Matrigel can increase the persistence of preexisting vessels by greater than threefold, with complex vessels remaining stable for more than 10 days. The results demonstrate that MSCs play an active role in the cellular processes involved in the formation, stabilization, and maturation of newly formed vessels. Further, these outcomes are not governed solely by either paracrine or direct contact effects and are both time and dose dependent.

The individuality of tumor-stroma interaction in non-small cell lung cancer: Insights from functional and molecular analyses

11115 Background: Tumor-stroma interaction plays a significant role for malignant growth. Results from prostate and breast cancer rodent models show cancerogenic properties of tumor-associated and genetically altered stromal cells (SC) when combined with initiated or normal epithelium (Olumi et al., Cancer Res 1999, Kuperwasser et al., PNAS 2004). However, data on the mechanisms and sequels of tumor-stroma interaction in lung cancer are scanty. Methods: Here, we analyzed the functional and molecular sequels of cross-talk between the non-small cell lung cancer (NSCLC) cell lines A549, H23, and H1703 and primary stromal cells (SC) derived from matched normal lung tissue and tumors of newly diagnosed NSCLC patients. Tumor cells were kept in a non-contact co-culture system with SC and analyzed for alterations in proliferation, colony formation, migration, adhesion, invasion, chemosensitivity and gene expression by Affymetrix HG U133 Plus 2.0 arrays. Results: Exposure to SC altered cellular functions and gene expression profiles related to tumor growth, metastasis and response to therapy. Each cell line showed individual alterations that were hierarchically governed by the (1) type of tumor cell, (2) the SC donor and his histology (3) and the local origin of the SC (normal lung tissue vs. tumor-associated). Conclusions: This in vitro model demonstrates an individual pattern of tumor-stroma interaction in NSCLC that is determined by both, the properties of the tumor cells and those of the stromal environment. Thus, biomarker programs in NSCLC should also consider the stromal compartment. No significant financial relationships to disclose.

Soluble factors from neocortical astrocytes enhance neuronal differentiation of neural progenitor cells from adult rat hippocampus on micropatterned polymer substrates

Rat adult hippocampal progenitor cells (AHPCs) are self-renewing, multipotent neural progenitors that have the ability to differentiate into neurons and glia. Previously, we demonstrated that coculture of AHPCs with postnatal day 2, type 1 cortical astrocytes on laminin-coated micropatterned polymer substrates facilitates selective neuronal differentiation of the AHPCs (Recknor et al., Biomaterials 2006;27:4098-4108). Under this condition, multidimensional cell-cell and/or cell-extracellular matrix interactions, as well as possible soluble factors released from astrocytes provided spatial and temporal control selectively enhancing neuronal differentiation and neurite alignment on topographically different regions of the same substrate. To investigate the potential role of astrocyte-derived soluble factors as cues involved in neuronal differentiation, a noncontact coculture system was used. Under control conditions, approximately 14% of the AHPCs were immunoreactive (IR) for the neuronal marker, class III beta-tubulin (TUJ1-IR). When cocultured in physical contact with astrocytes, neuronal differentiation increased significantly to about 25%, consistent with our previous results. Moreover, under noncontact coculture conditions using Transwell insert cultures, neuronal differentiation was dramatically increased to approximately 64%. Furthermore, neurite outgrowth from neuronal cell bodies was considerably greater on the patterned substrate when compared with the nonpatterned planar substrate under noncontact coculture conditions. Taken together, our results demonstrate that astrocyte-derived soluble factors provide cues for specific neuronal differentiation of AHPCs cultured on micropatterned substrates. In addition, a suppressive influence on neuronal differentiation appears to be mediated by contact with cocultured astrocytes. These results provide important insights into mechanisms for controlling neural progenitor/stem cell differentiation and facilitate development of strategies for CNS repair.