Hen-Li Chen

Mesenchymal stem cells promote growth and angiogenesis of tumors in mice

Though the early integration of mesenchymal stem cells (MSCs) into tumor-associated stroma of cancer has been demonstrated, the functional contributions and underlying mechanisms of these cells to tumor growth and angiogenesis remain to be clarified. Using a xenograft model, human colorectal cancer cells, MSCs, and their cell mixture were introduced to a subcutaneous site of immunodeficient mice. The tumor growth rate and angiogenesis of each transplantation was then compared. We demonstrate that a variety of colorectal cancer cells, when mixed with otherwise non-tumorigenic MSCs, increase the tumor growth rate and angiogenesis more than that when mixed with carcinoma-associated fibroblasts or normal colonic fibroblasts. The secretion of interleukin-6 (IL-6) from MSCs increases the secretion of endothelin-1 (ET-1) in cancer cells, which induces the activation of Akt and ERK in endothelial cells, thereby enhancing their capacities for recruitment and angiogenesis to tumor. The IL-6/ET-1/Akt or ERK pathway of tumor-stroma interaction can be targeted by an antibody against IL-6 or Lentiviral-mediated RNAi against IL-6 in MSCs, by inhibition or knockdown of ET-1 in cancer cells, or by inhibition of ERK and Akt in host endothelial cells. These demonstrate that attempts to interrupt the interaction of MSCs and cancer cells help to abrogate angiogenesis and inhibit tumor growth in tumors formed by cancer cells admixed with MSCs. These data demonstrate that the tumor microenvironment, namely, MSCs-secreted IL-6, may enrich the proangiognic factors secreted by cancer cells to increase angiogenesis and tumor growth and that targeting this interaction may lead to novel therapeutic and preventive strategies.

Enhancement of wound healing by human multipotent stromal cell conditioned medium: the paracrine factors and p38 MAPK activation.

Wound healing can be improved by transplanting mesenchymal stem cells (MSCs). In this study, we have demonstrated the benefits of the conditioned medium derived from human MSCs (CM-MSC) in wound healing using an excisional wound model. CM-MSC accelerated wound closure with increased reepithelialization, cell infiltration, granulation formation, and angiogenesis. Notably, CM-MSC enhanced epithelial and endothelial cell migration, suggesting the contribution of increased cell migration to wound healing enhanced by CM-MSC. Cytokine array, ELISA analysis, and quantitative RT-PCR revealed high levels of IL-6 in CM-MSC. Moreover, IL-6 added to the preconditioned medium enhanced both cell migration and wound healing, and antibodies against IL-6 blocked the increase in cell motility and wound closure by CM-MSC. The IL-6 secretory pathway of MSCs was inhibited by SB203580, an inhibitor of p38 MAPK or siRNA against p38 MAPK, suggesting IL-6 secretion by MSCs is mediated through the activation of p38 MAPK. Inactivation of p38 MAPK also reduced the expression and production of IL-8 and CXCL1 by MSCs, both of which were also demonstrated to enhance cell migration and wound closure. Thus, our data suggest MSCs promote wound healing through releasing a repertoire of paracrine factors via activation of p38 MAPK, and the CM-MSC may be applied to enhance wound healing.

Corneal repair by human corneal keratocyte-reprogrammed iPSCs and amphiphatic carboxymethyl-hexanoyl chitosan hydrogel

Induced pluripotent stem cells (iPSCs) have promising potential in regenerative medicine, but whether iPSCs can promote corneal reconstruction remains undetermined. In this study, we successfully reprogrammed human corneal keratocytes into iPSCs. To prevent feeder cell contamination, these iPSCs were cultured onto a serum- and feeder-free system in which they remained stable through 30 passages and showed ESC-like pluripotent property. To investigate the availability of iPSCs as bioengineered substitutes in corneal repair, we developed a thermo-gelling injectable amphiphatic carboxymethyl-hexanoyl chitosan (CHC) nanoscale hydrogel and found that such gel increased the viability and CD44+proportion of iPSCs, and maintained their stem-cell like gene expression, in the presence of culture media. Combined treatment of iPSC with CHC hydrogel (iPSC/CHC hydrogel) facilitated wound healing in surgical abrasion-injured corneas. In severe corneal damage induced by alkaline, iPSC/CHC hydrogel enhanced corneal reconstruction by downregulating oxidative stress and recruiting endogenous epithelial cells to restore corneal epithelial thickness. Therefore, we demonstrated that these human keratocyte-reprogrammed iPSCs, when combined with CHC hydrogel, can be used as a rapid delivery system to efficiently enhance corneal wound healing. In addition, iPSCs reprogrammed from corneal surgical residues may serve as an alternative cell source for personalized therapies for human corneal damage.

Knockdown of p21Cip1/Waf1 enhances proliferation, the expression of stemness markers, and osteogenic potential in human mesenchymal stem cells

Mammalian aging of many tissues is associated with a decline in the replicative and functional capacity of somatic stem cells. Understanding the basis of this decline is a major goal of aging research. Human bone marrow‐derived multipotent stromal cells (MSCs) have been applied in the treatment of fracture nonunion. Clinical application of MSCs requires abundant cells that can be overcome by ex vivo expansion of cells, but often at the expense of stemness and differentiation potentiality. We first demonstrated that late‐passage MSCs exhibited decreased proliferation capacity, reduced expression of stemness markers such as Oct‐4 and Nanog, and deterioration of osteogenic potential. Further, late‐passage MSCs showed increased expression of p21Cip1/Waf1 (p21), an inhibitor of the cyclin‐dependent kinase. Knockdown of p21 by lentivirus‐mediated shRNAs against p21 in late‐passage MSCs increased the proliferation capacity, the expression of Oct‐4 and Nanog, and osteogenic potential compared with cells transduced with control shRNA. More importantly, reduction in p21 expression in MSCs enhanced the bone repair capacity of MSCs in a rodent calvarial defect model. Knockdown of p21 in MSCs also increased the telomerase activity and telomere length, and did not show chromosomal abnormalities or acquire transformation ability. Therefore, these data successfully demonstrate the involvement of senescence gene in the expression of stemness markers and osteogenic potential of MSCs.

Combination of local and systemic parathyroid hormone enhances bone regeneration.

Parathyroid hormone is one of the most promising therapeutic agents for osteopororosis, but its use to facilitate bone regeneration in osseous defects is less clear. The purpose of the current study was to determine the effects of combining systematic parathyroid hormone and a local parathyroid hormone gene theraphy in a critical-sized osteotomy model. Rats received bilateral femoral osteotomies followed by implantation of a gene-activated matrix encoding parathyroid hormone (1–34) on one side and a control gene-activated matrix on te opposite side. Systematic parthyroid hormone (1–34) or vehicle was injected daily and rats were sacrificed 6 weeks later. Systematic parathyroid hormone increased bone mineral density and bone mineral content measured by dual-energy xray absorptiometry analysis of tibias and vertebrae, and increased serum osteocalcin levels during healing of osteotomies. Furthermore, comparing osteotomy sites that received the same gene-activated matrices as vehicle-injected rats, parathyroid hormone-injected rats showed trends of greater bone areas via histomorphometric and microdiographic analyses and higher osteocalcin mesenger ribonucleic acid expression via Northern blot analyses. The combination of systemic and local parathyroid hormone led to higher bone mineral density, bone mineral content, and bone area, a trend for greater radiographic-detected bone area and higher expression of osteocalcin in osteotomy sites when compared with the individual treatment or control groups. Local parathyroid hormone gene theraphy enhanced the anabolic effect of systemic parathyroid hormone during osteomy healing. This study supports the concept of a combined local and systemic approach for enhancing the repair of a fracture at risk for nonunion.

Improvement of carbon tetrachloride-induced acute hepatic failure by transplantation of induced pluripotent stem cells without reprogramming factor c-Myc.

The only curative treatment for hepatic failure is liver transplantation. Unfortunately, this treatment has several major limitations, as for example donor organ shortage. A previous report demonstrated that transplantation of induced pluripotent stem cells without reprogramming factor c-Myc (3-genes iPSCs) attenuates thioacetamide-induced hepatic failure with minimal incidence of tumorigenicity. In this study, we investigated whether 3-genes iPSC transplantation is capable of rescuing carbon tetrachloride (CCl4)-induced fulminant hepatic failure and hepatic encephalopathy in mice. Firstly, we demonstrated that 3-genes iPSCs possess the capacity to differentiate into hepatocyte-like cells (iPSC-Heps) that exhibit biological functions and express various hepatic specific markers. 3-genes iPSCs also exhibited several antioxidant enzymes that prevented CCl4-induced reactive oxygen species production and cell death. Intraperitoneal transplantation of either 3-genes iPSCs or 3-genes iPSC-Heps significantly reduced hepatic necrotic areas, improved hepatic functions, and survival rate in CCl4-treated mice. CCl4-induced hepatic encephalopathy was also improved by 3-genes iPSC transplantation. Hoechst staining confirmed the successful engraftment of both 3-genes iPSCs and 3-genes iPSC-Heps, indicating the homing properties of these cells. The most pronounced hepatoprotective effect of iPSCs appeared to originate from the highest antioxidant activity of 3-gene iPSCs among all transplanted cells. In summary, our findings demonstrated that 3-genes iPSCs serve as an available cell source for the treatment of an experimental model of acute liver diseases.

Intramarrow Bone Morphogenetic Protein 4 Gene Delivery Enhances Early Implant Stability in Femurs of Ovariectomized Rabbits

BACKGROUND Sufficient early implant stability is critical to prevent excessive micromovement of the implant during peri-implant healing and to ensure the success of osseointegration. Implants placed in osteoporotic bones are often associated with low early implant stability. The purpose of this study is to determine the effects of intramarrow bone morphogenetic protein 4 (BMP4) gene delivery on early implant stability and peri-implant healing. METHODS Adenoviruses encoding human BMP4 or LacZ were introduced into the femoral osteotomy sites immediately before implant placement in ovariectomized rabbits. The implant stability was determined by resonance frequency analysis at weeks 0, 4, and 8. Changes in cortical bone thickness and intrascrew bone formation at weeks 4 and 8 were evaluated by microcomputed tomography analysis and undecalcified histologic examination, respectively. RESULTS Intramarrow BMP4 gene delivery resulted in more improvement in implant stability at both weeks 4 and 8. Increased increment in peri-implant cortical bone thickness and better intrascrew bone formation were found in the BMP4 group compared to the LacZ group. CONCLUSION The results of this study suggest that intramarrow adenoviral gene delivery of BMP4 enhances peri-implant bone healing and improves early implant stability in osteoporotic rabbit femurs.

Intramarrow Bone Morphogenetic Protein 4 Gene Delivery Improves Local Bone Quality in Femurs of Ovariectomized Rabbits

BACKGROUND Poor bone quality at implant recipient site is a major risk factor for implant failure. The purpose of this study is to examine the potential of intramarrow bone morphogenetic protein 4 (BMP4) gene delivery for local bone quality improvement. METHODS Adenoviral vector encoding human BMP4 (Ad-BMP4) was constructed. Adenovirus encoding β-galactosidase (Ad-LacZ) was used as a control virus. Ad-BMP4 and Ad-LacZ were injected into femurs of ovariectomized rabbits. The temporal changes in bone mineral density at injected areas were determined by repeated measurements by dual-energy x-ray absorptiometry at 0, 1, 2, 4, and 8 weeks after injection. The effects of gene delivery on cortical bone and cancellous bone were evaluated by microcomputed tomography analysis and histologic examination at 8 weeks. RESULTS The bone mineral density of the BMP4 group was significantly higher than the LacZ group at 4 and 8 weeks by 61% and 35%, respectively. Results from microcomputed tomography analysis and histologic examination at 8 weeks indicated thicker cortical bone and denser cancellous bone in the BMP4 group compared to the LacZ group. CONCLUSIONS Intramarrow gene delivery of BMP4 effectively improved local bone quality for at least 8 weeks. The sustained delivery of osteogenic factors via local gene therapy approach may reduce implant failures associated with poor local bone quality.

Effect of circumferential undercut placement in the root canal wall on the retention of a tapered post in large and small root canals

OBJECTIVES This study examined the influence of root canal space and circumferential undercut placement in the dentine wall on the retention of a prefabricated Root Keeper post. METHODS Twenty-four extracted single-rooted teeth, having narrow root canal spaces, were decoronated, treated endodontically, and then centrally embedded in resin blocks. All canal spaces were prepared with a no. 2 Peeso reamer to an 8mm depth, and half the specimens were successively treated with a no. 3 Peeso to depths of 5 and 8mm for the small canal group (SC) and large canal group (LC), respectively. An L-type keeper post (Aichi Steel Co.) was luted to each canal with resin cement. The assembly was thermocycled 2000 times (4-60 degrees C), and post retention was measured. Afterwards the dislodged post with the intact structure was sandblasted, and the canal was cleaned and slightly undercut using an inverted cone bur. Six treated posts were recemented for both grooved large canal (GLC) and grooved small canal (GSC) groups, and the repaired retentive strengths were measured. RESULTS Keeper posts cemented in SC (84.26+/-31.85N) showed better (p<0.05) retention than those cemented in LC (41.92+/-22.36N). Interestingly, the repaired strengths of the recemented post increased in both GSC (104.29+/-11.89N) and GLC (96.65+/-6.91N). All posts, except for one in the GLC, failed at the keeper-post joint. CONCLUSIONS Prefabricated Root Keeper posts had lower retentive strength in roots with a large canal space. Placement of a circumferential undercut in the root canal wall significantly enhanced the retention of the short tapered posts.

Promoting Induced Pluripotent Stem Cell-driven Biomineralization and Periodontal Regeneration in Rats with Maxillary-Molar Defects using Injectable BMP-6 Hydrogel

Periodontal disease may cause considerable destruction of alveolar bone, periodontal ligaments (PDLs) and cementum and even lead to progressive oral dysfunction. Periodontal tissue regeneration is the ultimate goal of periodontal disease treatment to reconstruct both structures and functions. However, the regenerative efficiency is low, possibly due to the lack of a proper periodontal microenvironment. In this study, we applied an injectable and thermosensitive chitosan/gelatin/glycerol phosphate hydrogel to provide a 3D environment for transplanted stem cells and to enhance stem cell delivery and engraftment. The iPSCs-BMP-6-hydrogel complex promoted osteogenesis and the differentiation of new connective tissue and PDL formation. In animal models of maxillary-molar defects, the iPSCs-BMP-6-hydrogel-treated group showed significant mineralization with increased bone volume, trabecular number and trabecular thickness. Synergistic effects of iPSCs and BMP-6 increased both bone and cementum formation. IPSCs-BMP-6-hydrogel-treated animals showed new bone synthesis (increased ALP- and TRAP-positive cells), new PDL regeneration (shown through Masson’s trichrome staining and a qualification assay), and reduced levels of inflammatory cytokines. These findings suggest that hydrogel-encapsulated iPSCs combined with BMP-6 provide a new strategy to enhance periodontal regeneration. This combination not only promoted stem cell-derived graft engraftment but also minimized the progress of inflammation, which resulted in highly possible periodontal regeneration.