Win Ping Deng

Site-directed mutagenesis of virtually any plasmid by eliminating a unique site

We describe an efficient site-specific mutagenesis procedure that is effective with virtually any plasmid, requiring only that the target plasmid carry a unique, nonessential restriction site. The procedure employs two mutagenic oligonucleotide primers. One primer contains the desired mutation and the second contains a mutation in any unique, nonessential restriction site. The two primers are annealed to circular single-stranded DNA (produced by heating circular double-stranded DNA) and direct synthesis of a new second strand containing both primers. The resulting DNA is transformed into a mismatch repair defective (mut S) Escherichia coli strain, which increases the probability that the two mutations will cosegregate during the first round of DNA replication. Transformants are selected en masse in liquid medium containing an appropriate antibiotic and plasmid DNA is prepared, treated with the enzyme that recognizes the unique, nonessential restriction site, and retransformed into an appropriate host. Linearized parental molecules transform bacteria inefficiently. Plasmids with mutations in the unique restriction site are resistant to digestion, remain circular, and transform bacteria efficiently. By linking a selectable mutation in a unique restriction site to a nonselectable mutation, the latter can be recovered at frequencies of about 80%. Since most plasmids share common vector sequences, few primers, targeted to shared restriction sites, are needed for mutagenizing virtually any plasmid. The procedure employs simple procedures, common materials, and it can be performed in as little as 2 days.

Mesenchymal Stem Cell Targeting of Microscopic Tumors and Tumor Stroma Development Monitored by Noninvasive In vivo Positron Emission Tomography Imaging

The aim of this study was to assess the efficacy human mesenchymal stem cells (hMSC) for targeting microscopic tumors and suicide gene or cytokine gene therapy. Immunodeficient mice were transplanted s.c. with human colon cancer cells of HT-29 Inv2 or CCS line, and 3 to 4 days later, i.v. with “tracer” hMSCs expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK) and enhanced green fluorescent protein (EGFP) reporter genes. Subsequently, these tumors were examined for specificity and magnitude of HSV1-TK+, EGFP+ stem cell engraftment and proliferation in tumor stroma by in vivo positron emission tomography (PET) with 18F-labeled 9-(4-fluoro-3-hydroxymethylbutyl)-guanine ([18F]-FHBG). In vivo PET images of tumors growing for 4 weeks showed the presence of HSV1-TK+ tumor stroma with an average of 0.36 ± 0.24% ID/g [18F]-FHBG accumulation. In vivo imaging results were validated by in situ correlative histochemical, immunofluorescent, and cytometric analyses, which revealed EGFP expression in vWF+ and CD31+ endothelial cells of capillaries and larger blood vessels, in germinal layer of dermis and hair follicles proximal to the s.c. tumor site. These differentiated HSV1-TK+, GFP+ endothelial cells had limited proliferative capacity and a short life span of <2 weeks in tumor fragments transplanted into secondary hosts. We conclude that hMSCs can target microscopic tumors, subsequently proliferate and differentiate, and contribute to formation of a significant portion of tumor stroma. PET imaging should facilitate clinical translation of stem cell–based anticancer gene therapeutic approaches by providing the means for in vivo noninvasive whole-body monitoring of trafficking, tumor targeting, and proliferation of HSV1-tk-expressing “tracer” hMSCs in tumor stroma.

In vitro stage-specific chondrogenesis of mesenchymal stem cells committed to chondrocytes

OBJECTIVE Osteoarthritis is characterized by an imbalance in cartilage homeostasis, which could potentially be corrected by mesenchymal stem cell (MSC)-based therapies. However, in vivo implantation of undifferentiated MSCs has led to unexpected results. This study was undertaken to establish a model for preconditioning of MSCs toward chondrogenesis as a more effective clinical tool for cartilage regeneration. METHODS A coculture preconditioning system was used to improve the chondrogenic potential of human MSCs and to study the detailed stages of chondrogenesis of MSCs, using a human MSC line, Kp-hMSC, in commitment cocultures with a human chondrocyte line, hPi (labeled with green fluorescent protein [GFP]). In addition, committed MSCs were seeded into a collagen scaffold and analyzed for their neocartilage-forming ability. RESULTS Coculture of hPi-GFP chondrocytes with Kp-hMSCs induced chondrogenesis, as indicated by the increased expression of chondrogenic genes and accumulation of chondrogenic matrix, but with no effect on osteogenic markers. The chondrogenic process of committed MSCs was initiated with highly activated chondrogenic adhesion molecules and stimulated cartilage developmental growth factors, including members of the transforming growth factor beta superfamily and their downstream regulators, the Smads, as well as endothelial growth factor, fibroblast growth factor, insulin-like growth factor, and vascular endothelial growth factor. Furthermore, committed Kp-hMSCs acquired neocartilage-forming potential within the collagen scaffold. CONCLUSION These findings help define the molecular markers of chondrogenesis and more accurately delineate the stages of chondrogenesis during chondrocytic differentiation of human MSCs. The results indicate that human MSCs committed to the chondroprogenitor stage of chondrocytic differentiation undergo detailed chondrogenic changes. This model of in vitro chondrogenesis of human MSCs represents an advance in cell-based transplantation for future clinical use.

Tissue-engineered intervertebral disc and chondrogenesis using human nucleus pulposus regulated through TGF-β1 in platelet-rich plasma

Human intervertebral disc (IVD) degeneration often initiated from the human nucleus pulposus (hNP) with aging leading to IVD destruction and extracellular matrix (ECM) depletion. Previously, we have successfully employed transforming growth factor‐β1 (TGF‐β1) to promote chondrogenesis of mesenchymal progenitor cells (MPCs) and immortalized human mesenchymal stem cells. In this study, we examine the role of TGF‐β1 in platelet‐rich plasma (PRP) on disc regeneration, including proliferation, redifferentiation, and the reconstitution of tissue‐engineered NP. hNP cells were isolated from volunteers with different ages and cultured in the presence of PRP. We found that the most effective concentration for hNP proliferation was 1 ng/ml TGF‐β1 in PRP, which was further applied in the following experiments. hNP cell proliferation in all age groups were increased time‐dependently by PRP and cell morphologies showed aggregation. The mRNA of Sox9, type II collagen, and aggrecan were all significantly upregulated by PRP through RT‐PCR. Glycosaminoglycan (GAG) accumulation reached the highest value at day 7 and continued to day 9 culture. PRP promoted NP regeneration via the Smad pathway was also determined and highly activated p‐Smad2/3 at 30 min and continuously sustained to 120 min. Immunostaining of type II collagen indicates that PRP participates in chondrogenesis of tissue‐engineered NP with collagen scaffolds. We concluded that growth factors in PRP can effectively react as a growth factor cocktail to induce hNP proliferation and differentiation, and also promote tissue‐engineered NP formation. These findings are the first to demonstrate that PRP might be a therapeutic candidate for prevention of disc degeneration. J. Cell. Physiol. 209: 744–754, 2006.

Intervertebral disc regeneration in an ex vivo culture system using mesenchymal stem cells and platelet-rich plasma

An ex vivo degenerative intervertebral disc (IVD) organ culture system was established for the screening of disc regeneration agents. Its application was demonstrated by a stem cell and growth factor-based therapeutic approach for the amelioration of IVD. An ex vivo culture system using chymopapain to partially digest nucleus proposus tissue was established to mimic human IVD degeneration. This system was then used for the evaluation of different therapeutic regimens including: mesenchymal stem cell derived from eGFP-transgenic porcine (MSC-GFP), platelet-rich plasma (PRP) and MSC-GFP/PRP combined treatment, and confirmed in in vivo animal model. Chondrogenic-specific gene products including Col II and aggrecan were found upregulated and chondrogenic matrix deposition increased, as evident by sustained fluorescent signals over 4 weeks, in the MSC-GFP implanted group. Previously, we demonstrated in vitro stage-specific chondrogenesis of MSC by chondrocytic commitment. These same molecules upregulated for chondrogenesis were also observed in MSC-GFP group. PRP that has been shown to promote nucleus pulposus (NP) regeneration also resulted in significant increased levels of mRNA involved in chondrogenesis and matrices accumulation. The ex vivo IVD regeneration results were repeated and supported by in vivo porcine degenerative system. Moreover, the disc height index (DHI) was significantly increased in both in vivo MSC-GFP and PRP regeneration groups. Unexpectedly, the MSC-GFP/PRP combined therapy demonstrated an inclination towards osteogenesis in ex vivo system. The ex vivo degenerative IVD culture system described in this study could serve as an alternative and more accessible model over large animal model. This system also provides a high-throughput platform for screening therapeutic agents for IVD regeneration.

The balance between adipogenesis and osteogenesis in bone regeneration by platelet-rich plasma for age-related osteoporosis

The aim of this study was to develop a new diagnostic and therapeutic approach for the treatment of osteoporosis. Previously, we demonstrated that intraosseous transplantation of platelet-rich plasma (PRP) treated-osteoblast-like cells into ovariectomized senescence-accelerated mice (OVX-SAMP8) prevented the development of osteoporosis. In continuation, we aimed to explore the complex etiology of osteoporosis using this platform. An inverse relationship between bone marrow adipogenesis and osteogenesis has been suggested in the development of osteoporosis but the underlying mechanisms remain poorly described. To address these issues, we used PRP to inhibit adipocyte differentiation by promoting osteoblastic differentiation in adipocytes. In addition, a positive correlation between an increase in bone marrow adipocytes and bone loss was established. We assessed this relationship using an osteoporotic animal disease model which consisted of young (for prevention) and old (for treatment) OVX-SAMP8 mice. This animal model demonstrated that PRP treatment mainly exerted its action via promoting bone regeneration but also appeared to suppress adipogenesis within the marrow. The findings and methodology of this study could potentially be applied in the prevention and treatment of osteoporosis.

Regenerative potentials of platelet-rich plasma enhanced by collagen in retrieving pro-inflammatory cytokine-inhibited chondrogenesis

This study was undertaken to evaluate the role of collagen matrix to enhance platelet-rich plasma (PRP) effects on pro-inflammatory cytokine-induced arthritic model. We have previously demonstrated the highly regenerative roles of PRP to restore disc degeneration and osteoporosis. In this study, PRP modulated by collagen matrix was used as a regenerative and anti-inflammatory mediator to rescue the chondrocyte degeneration induced by pro-inflammatory cytokines IL-1β (10 ng/ml)+TNF-α (20 ng/ml). First, the MTT result indicated that 1 ng/ml TGF-β1 in PRP showed an optimal dosage for chondrocytes proliferation. The chondrogenic-specific gene expressions were rescued by PRP from the inhibition of IL-1β+TNF-α, especially under the modulation of collagen matrix. The inflammatory molecules activated by IL-1β+TNF-α were also significantly diminished by PRP with collagen matrix. The membrane receptors integrin α1β1 and CD44 were strongly inhibited by IL-1β+TNF-α, while this inhibition was then recovered by PRP in collagen coating condition. In a 3D model encapsulated with collagen, PRP-induced chondrogenesis were highly enhanced, such as strong restoration of type II collagen and proteoglycan from the inhibition of IL-1β+TNF-α. The result indicated that collagen matrix enhances the effect of PRP on chondrogenesis in response to pro-inflammatory cytokines. The combination of PRP and collagen matrix might facilitate a physiological microenvironment beneficial for maintaining chondrocyte homeostasis and represents an advanced osteoarthritis therapy for clinical applications.

Infrared thermography to mass-screen suspected sars patients with fever

Fever greater than 38°C is a cardinal sign of patients with the severe acute respiratory syndromes (SARS). To reduce the risk of nosocomial cross infections, screening all patients and visitors who visit hospitals and clinics for fever at the entrance of every hospital building has become a standard protocol in Taiwan during the SARS epidemic from mid-April to mid-June 2003. We used a digital infrared thermal imaging (DITI) system (Telesis Spectrum 9000 MB) to conduct mass screening of patients and visitors who entered the hospital to identify those with fever. The DITI system has two components: a sensor head and a PC imaging workstation. The sensor head is an optic-mechanical device which consists of imagining optics for focusing the infrared source information on the infrared detector. The infrared images are further converted into electrical signals, which are then processed for real-time display on the monitor. During the period from April 13 to May 12 2003, 72,327 outpatients and visitors entered Taipei Medical University-Wan Fang Hospital, Taipei, Taiwan. A total of 305 febrile patients (0.42%) was detected by infrared thermography. Among them, three probable SARS patients were identified after thorough studies including contact history, laboratory tests and radiology examinations. The findings suggests that infrared thermography was an effective and reliable tool ideal for mass-screening patients with fever in the initial phase of screening for SARS patients at a busy hospital which sees approximately 3,000 outpatients every weekday during the SARS epidemic. Asia Pac J Public Health 2005: 17(1): 26-28.

Synergistic anabolic actions of hyaluronic acid and platelet-rich plasma on cartilage regeneration in osteoarthritis therapy

Osteoarthritis (OA) is a common disease associated with tissue inflammation, physical disability and imbalanced homeostasis in cartilage. For advanced treatments, biological approaches are currently focused on tissue regeneration and anti-inflammation. This study was undertaken to evaluate the therapeutic efficacies of hyaluronic acid (HA) and platelet-rich plasma (PRP) (HA+PRP) on OA. Articular chondrocytes were obtained from five OA patients. The optimal HA and PRP concentrations were evaluated by MTT assay. The expressions of chondrogenic and inflammatory genes were analyzed by RT-PCR. Signaling pathway was examined by immunoblotting and the expressions of OA pathology-related chemokines and cytokines was demonstrated by real-time PCR-based SuperArray. The therapeutic efficacies of HA+PRP were then demonstrated in 3D arthritic neo-cartilage and ACLT-OA model. Here we showed that HA+PRP could greatly retrieve pro-inflammatory cytokines-reduced articular chondrocytes proliferation and chondrogenic phenotypes, the mechanism of which involve the sequential activation of specific receptors CD44 and TGF-βRII, downstream mediators Smad2/3 and Erk1/2, and the chondrogenic transcription factor SOX9. The real-time PCR-based SuperArray results also indicated that OA pathology-related chemokines and cytokines could be efficiently suppressed by HA+PRP. Moreover, the cartilaginous ECM could be retrieved from inflammation-induced degradation by HA+PRP in both 2D monolayer and 3D neo-cartilage model. Finally, the intra-articular injection of HA+PRP could strongly rescue the meniscus tear and cartilage breakdown and then decrease OA-related immune cells. The combination of HA+PRP can synergistically promote cartilage regeneration and inhibit OA inflammation. This study might offer an advanced and alternative OA treatment based on detailed regenerative mechanisms.

Identification of Antrocin from Antrodia camphorata as a Selective and Novel Class of Small Molecule Inhibitor of Akt/mTOR Signaling in Metastatic Breast Cancer MDA-MB-231 Cells

The PI3K/Akt/mTOR pathway is considered to be an attractive target for the development of novel anticancer molecules. This paper reports for the first time that a small molecule, antrocin (MW = 234), from Antrodia camphorata was a potent antagonist in various cancer types, being highest in metastatic breast cancer MDA-MB-231 cells (MMCs) with an IC(50) value of 0.6 μM. Antrocin was a superior antiproliferator in MMCs as compared with doxorubicin and cisplatin, prevents colony formation, and was nontoxic to nontumorgenic MCF10A and HS-68 cells. Antrocin induced dose-dependent apoptosis in MMCs and caused cleavage of caspase-3 and poly(ADP-ribose) polymerase. Antrocin also caused a time-dependent decrease in protein expression of anti-apoptotic Bcl-2, Bcl-xL, survivin, and their mRNA, with concomitant increase in pro-apoptotic Bax and cytosolic cytochrome c. In a mechanistic study, antrocin suppressed the phosphorylation of Akt and its downstream effectors mTOR, GSK-3β, and NF-κB. Furthermore, down-regulation of Akt by small interfering RNA prior to antrocin treatment resulted in enhanced cell growth inhibition and apoptosis. Thus, antrocin as an Akt/mTOR dual inhibitor has broad applicability in the development of a clinical trial candidate for the treatment of metastatic breast cancer.