Nien-Hsien Liou

siRNA-targeting transforming growth factor-β type I receptor reduces wound scarring and extracellular matrix deposition of scar tissue.

Hypertrophic scarring is related to persistent activation of transforming growth factor-β (TGF-β)/Smad signaling. In the TGF-β/Smad signaling cascade, the TGF-β type I receptor (TGFBRI) phosphorylates Smad proteins to induce fibroblast proliferation and extracellular matrix deposition. In this study, we inhibited TGFBRI gene expression via TGFBRI small interfering RNA (siRNA) to reduce fibroblast proliferation and extracellular matrix deposition. Our results demonstrate that downregulating TGFBRI expression in cultured human hypertrophic scar fibroblasts significantly suppressed cell proliferation and reduced type I collagen, type III collagen, fibronectin, and connective tissue growth factor (CTGF) mRNA, and type I collagen and fibronectin protein expression. In addition, we applied TGFBRI siRNA to wound granulation tissue in a rabbit model of hypertrophic scarring. Downregulating TGFBRI expression reduced wound scarring, the extracellular matrix deposition of scar tissue, and decreased CTGF and α-smooth muscle actin mRNA expression in vivo. These results suggest that TGFBRI siRNA could be applied clinically to prevent hypertrophic scarring.

Validation of 4-[18F]-ADAM as a SERT imaging agent using micro-PET and autoradiography.

Serotonin transporters (SERTs) have been implicated in various neuropsychiatric disorders. We aim to validate 4-[(18)F]-ADAM (N,N-dimethyl-2-(2-amino-4-[(18)F]fluorophenylthio)benzylamine) as a SERT imaging agent in rats using micro-positron emission tomography (micro-PET) and autoradiography. Sixty to ninety min after injecting 4-[(18)F]-ADAM, specific uptake ratios (SURs) were determined by micro-PET measurements in various brain regions of normal control rats. For n=3, the SUR in the midbrain was 4.94+/-0.16, for the hypothalamus it was 4.39+/-0.031 and for the caudate it was 4.18+/-0.53. The retention of 4-[(18)F]-ADAM in the hypothalamus and midbrain regions increased rapidly between 5 to 10 min after injection and declined thereafter. The SURs determined by autoradiography were: 9.31+/-1.41 for the midbrain, 7.15+/-1.45 for the hypothalamus and 5.22+/-1.14 for the caudate putamen. Both micro-PET and autoradiography studies revealed a dose-dependent progressive inhibition of radioligand uptake in the frontal cortex, caudate putamen and hypothalamus in rats treated with 0.01 to 0.25 mg/kg paroxetine. A decrease in 4-[(18)F]-ADAM uptake of approximately 84% was observed in the midbrain of rats pretreated with 0.25 mg/kg paroxetine as compared to controls (4.94+/-0.16 versus 0.80+/-0.17, n=3). Both 5,7-dihydroxytryptamine and p-chloroamphetamine-treated rats showed pronounced reduction in 4-[(18)F]-ADAM binding when compared to normal controls. Rats pretreated with p-chloroamphetamine exhibited significant inhibition of 4-[(18)F]-ADAM uptake in brain regions rich in SERT over a period of four weeks. Thus, 4-[(18)F]-ADAM is a SERT-specific radioligand that may be useful for evaluating neuropsychiatric conditions involving serotonergic dysfunction.

Effects of interleukin-15 on neuronal differentiation of neural stem cells.

Interleukin-15 (IL-15) signaling has pleiotropic actions in many cell types during development and has been best studied in cells of immune system lineage, where IL-15 stimulates proliferation of cytotoxic T cells and induces maturation of natural killer cells. A few reports have indicated that IL-15 and the IL-15 receptor are expressed in central nervous system tissues and neuronal cell lines. Because this aspect of IL-15 action is poorly studied, we used cultured rat neural stem cells (NSCs) to study IL-15 signal transduction and activity. Primary cultures of rat NSCs in culture will form neurospheres and will differentiate into neuron, astrocyte, and oligodendrocyte progenitors under permissive conditions. We found by immunofluorescence that the IL-15Ralpha subunit of the IL-15 receptor was expressed in NSCs and differentiating neurons, but not astrocyte or oligodendrocyte progenitors. We also showed that IL-15 treatment reduced MAP-2 protein levels in neurons and could reduce neurite outgrowth in differentiating neurons but did not affect NSC proliferation, and cell proportions and viability of the corresponding lineage cells. In the presence of a STAT3 inhibitor, Stattic, IL-15 no longer reduced MAP-2 protein levels. IL-15 treatment caused STAT3 phosphorylation. Furthermore, using anti-IL-15Ralpha antibody to block IL-15 signaling completely inhibited IL-15-induced phosphorylation of STAT3 and prevented IL-15 from decreasing neurite outgrowth. In conclusion, IL-15 may influence neural cell differentiation through a signal transduction pathway involving IL-15Ralpha and STAT3. This signal transduction modifies MAP-2 protein levels and, consequently, the differentiation of neurons from NSCs, as evidenced by reduced neurite outgrowth.

The effects of microenvironment on wound healing by keratinocytes derived from mesenchymal stem cells

AbstractEmbryonic stem cells (ESCs) are pluripotent cells that can differentiate into various cell types, including keratinocyte-like cells, within suitable microniches. In this study, we aimed to investigate the effects of culture media, cell coculture, and a tissue-engineering biocomposite on the differentiation of mouse ESCs (MESCs) into keratinocyte-like cells and applied these cells to a surgical skin wound model. MESCs from BALB/c mice (ESC26GJ), which were transfected using pCX-EGFP expressing green fluorescence, were used to track MESC-derived keratinocytes. Weak expression of the keratinocyte early marker Cytokeratin 14 (CK-14) was observed up to 12 days when MESCs were cultured in a keratinocyte culture medium on tissue culture plastic and on a gelatin/collagen/polycaprolactone (GCP) biocomposite. MESCs cocultured with human keratinocyte cells (HKCs) also expressed CK-14, but did not express CK-14 when cocultured with human fibroblast cells (HFCs). Furthermore, CK-14 expression was observed when MESCs were cocultured by seeding HKCs or HFCs on the same or opposite side of the GCP biocomposite. The highest CK-14 expression was observed by seeding MESCs and HKCs on the same side of the GCP composite and with HFCs on the opposite side. To verify the effectiveness of wound healing in vivo, adipose-derived stem cells were applied to treat surgical wounds in nude mice. An obvious epidermis multilayer and better collagen deposition during wound healing were observed, as assessed by Masson staining. This study demonstrated the potential of keratinocyte-like differentiation from mesenchymal stem cells for use in promoting wound closure and skin regeneration.

Osteogenic prospective of deriving human dental stem cells in collagen matrix boost

Stem cells derived from oral tissue represent a highly attractive alternative source for clinical bone regeneration because they can be collected by non-invasive or minimally invasive procedures. Herein, we describe the human dental stem cells (DSCs) deriving from buccal fat pads (BFP), dental pulp (DP) of impacted teeth, and periodontal ligaments (PDL) to obtain BFPSCs, DPSCs, and PDLSCs, respectively. Cells were purified with selected medium and expanded through passages in stem cell culture medium. Purified cells were characterized for stemness by their growth rate, immunostaining, and multilineage differentiation ability. They showed plastic adherence, expression of stemness-specific markers, and multilineage differentiation potential. Immunocytochemistry analysis confirmed that DPSCs had more osteogenic potential than BFSCs and PDLSCs. Calcium-rich deposits, evaluated by von Kossa and Alizarin red staining, showed greater mineralization when DPSCs were cultured on collagen type I matrix than without collagen. Furthermore, DPSC-seeded collagen type I matrix maintained consistent osteogenesis and boosted mineral formation by 1–2 weeks over that in DPSCs cultured without collagen. Radiographic analysis of DPSC-seeded collagen type I matrix transplanted into rat cranial defects showed significant bone regeneration after 8 weeks. These results suggested that the redundant oral tissue can be used as a source of adult multipotent stem cells for clinical bone regeneration.Graphical abstractTriple overlay images with biomarkers (red), nuclei (blue) and bright field morphology of DPSCs. The specifically osteo-differentiation shown by osteocalcin (left) expression and lack of sox9 (right) expressed in the images below which were cultured with collagen matrix, contrast with no collagen matrix group above.

Transneuronal Degeneration of Thalamic Nuclei following Middle Cerebral Artery Occlusion in Rats

Objective. Postinfarction transneuronal degeneration refers to secondary neuronal death that occurs within a few days to weeks following the disruption of input or output to synapsed neurons sustaining ischemic insults. The thalamus receives its blood supply from the posterior circulation; however, infarctions of the middle cerebral arterial may cause secondary transneuronal degeneration in the thalamus. In this study, we presented the areas of ischemia and associated transneuronal degeneration following MCAo in a rat model. Materials and Methods. Eighteen 12-week-old male Sprague-Dawley rats were randomly assigned to receive middle cerebral artery occlusion surgery for 1, 7, and 14 days. Cerebral atrophy was assessed by 2,3,5-triphenyltetrazolium hydrochloride staining. Postural reflex and open field tests were performed prior to animal sacrifice to assess the effects of occlusion on behavior. Results. Myelin loss was observed at the lesion site following ischemia. Gliosis was also observed in thalamic regions 14 days following occlusion. Differential degrees of increased vascular endothelial growth factor expression were observed at each stage of infarction. Increases in myelin basic protein levels were also observed in the 14-day group. Conclusion. The present rat model of ischemia provides evidence of transneuronal degeneration within the first 14 days of occlusion. The observed changes in protein expression may be associated with self-repair mechanisms in the damaged brain.

High correlation between skin color based on CIELAB color space, epidermal melanocyte ratio, and melanocyte melanin content

Background To treat skin color disorders, such as vitiligo or burns, melanocytes are transplanted for tissue regeneration. However, melanocyte distribution in the human body varies with age and location, making it difficult to select the optimal donor skin to achieve a desired color match. Determining the correlations with the desired skin color measurement based on CIELAB color, epidermal melanocyte numbers, and melanin content of individual melanocytes is critical for clinical application. Method Fifteen foreskin samples from Asian young adults were analyzed for skin color, melanocyte ratio (melanocyte proportion in the epidermis), and melanin concentration. Furthermore, an equation was developed based on CIELAB color with melanocyte ratio, melanin concentration, and the product of melanocyte ratio and melanin concentration. The equation was validated by seeding different ratios of keratinocytes and melanocytes in tissue-engineered skin substitutes, and the degree of fitness in expected skin color was confirmed. Results Linear regression analysis revealed a significant strong negative correlation (r = − 0.847, R2 = 0.717) between CIELAB L* value and the product of the epidermal melanocyte ratio and cell-based melanin concentration. Furthermore, the results showed that an optimal skin color match was achieved by the formula. Discussion We found that L* value was correlated with the value obtained from multiplying the epidermal melanocyte ratio (R) and melanin content (M) and that this correlation was more significant than either L* vs M or L* vs R. This suggests that more accurate prediction of skin color can be achieved by considering both R and M. Therefore, precise skin color match in treating vitiligo or burn patients would be potentially achievable based on extensive collection of skin data from people of Asian descent.

Correction to: Osteogenic prospective of deriving human dental stem cells in collagen matrix boost

The original version of this article unfortunately contained a mistake. The country was incorrect in the authors affiliations. It should read as “ROC”. The corrected affiliations are given below.

Histochemical and functional improvement of adipose-derived stem cell-based tissue-engineered cartilage by hyperbaric oxygen/air treatment in a rabbit articular defect model.

AbstractCartilage is exposed to compression forces during joint loading. Therefore, exogenous stimuli are frequently used in cartilage tissue engineering strategies to enhance chondrocyte differentiation and extracellular matrix (ECM) secretion. In this study, human adipose-derived stem cells were seeded on a gelatin/polycaprolactone scaffold to evaluate the histochemical and functional improvement of tissue-engineered cartilage after hyperbaric oxygen/air treatment in a rabbit articular defect model. Behavior tests showed beneficial effects on weight-bearing and rear leg-supporting capacities after treatment of tissue-engineered cartilage with 2.5 ATA oxygen or air. Moreover, positron emission tomography images and immunohistochemistry staining demonstrated hydroxyapatite formation and increased ECM synthesis, respectively, at the tissue-engineered cartilage graft site after high pressure oxygen/air treatment. Based on these results, we concluded that hyperbaric oxygen and air treatment can improve the quality of tissue-engineered cartilage in vivo by increasing the synthesis of ECM.