Construction of tissue-engineered lymphatic vessel using human adipose derived stem cells differentiated lymphatic endothelial like cells and decellularized arterial scaffold: A preliminary study.

We have previously demonstrated that human adipose-derived stem cells (hADSCs) can be differentiated into lymphatic endothelial like cells. The purpose of this study was to investigate the feasibility of utilizing the induced lymphatic endothelial like cells and decellularized arterial scaffold to construct the tissue-engineered lymphatic vessel. The hADSCs were isolated from adipose tissue in healthy adults and were characterized the multilineage differentiation potential. Decellularized arterial scaffold was prepared using the Triton x-100 method. ADSCs were differentiated into lymphatic-like endothelial cells, and the induced cells were then seeded onto the decellularized arterial scaffold to engineer the lymphatic vessel. The histological analyses were performed to examine the endothelialized construct. The decellularized arterial scaffold was successfully obtained and was able to maintain its vessel morphology. The isolated ADSCs can be differentiated into osteocytes and adipocytes. After seeding onto the scaffold, the seeded cells attached and grew well on the decellularized arterial scaffold. Our preliminary results demonstrated that the induced lymphatic endothelial like cells combined with decellularized arterial scaffold could be utilized to successfully engineer the lymphatic vessel. Our findings may be helpful for the development of tissue-engineering of the lymphatic graft.

Transcriptome Analysis of Blunt Snout Bream (Megalobrama amblycephala) Reveals Putative Differential Expression Genes Related to Growth and Hypoxia.

The blunt snout bream (Megalobrama amblycephala) is an important freshwater aquaculture species, but it is sensitive to hypoxia. No transcriptome data related to growth and hypoxia response are available for this species. In this study, we performed de novo transcriptome sequencing for the liver and gills of the fast-growth family and slow-growth family derived from 'Pujiang No.1' F10 blunt snout bream that were under hypoxic stress and normoxia, respectively. The fish were divided into the following 4 groups: fast-growth family under hypoxic stress, FH; slow-growth family under hypoxic stress, SH; fast-growth family under normoxia, FN; and slow-growth family under normoxia, SN. A total of 185 million high-quality reads were obtained from the normalized cDNA of the pooled samples, which were assembled into 465,582 contigs and 237,172 transcripts. A total of 31,338 transcripts from the same locus (unigenes) were annotated and assigned to 104 functional groups, and 23,103 unigenes were classified into seven main categories, including 45 secondary KEGG pathways. A total of 22,255 (71%) known putative unigenes were found to be shared across the genomes of five model fish species and mammals, and a substantial number (9.4%) of potentially novel genes were identified. When 6,639 unigenes were used in the analysis of differential expression (DE) genes, the number of putative DE genes related to growth pathways in FH, SH, SN and FN was 159, 118, 92 and 65 in both the liver and gills, respectively, and the number of DE genes related to hypoxic response was 57, 33, 23 and 21 in FH, FN, SH and SN, respectively. Our results suggest that growth performance of the fast-growth family should be due to complex mutual gene regulatory mechanisms of these putative DE genes between growth and hypoxia.

Two follistatin-like 1 homologs are differentially expressed in adult tissues and during embryogenesis in grass carp (Ctenopharyngodon idellus).

Follistatin-like 1 (Fstl1) peptides play important roles in inhibiting myoblast proliferation and differentiation. Here, we characterized and examined the expression patterns of fstl1a and -b in grass carp (Ctenopharyngodon idellus). These genes encode 314 aa and 310 aa peptides, respectively, sharing a sequence identity of 83%. Except for the existence of the follistatin-N-terminal (FOLN) and Kazal-type 2 serine protease inhibitor (Kazal 2) domains, grass carp Fstl1a and -b do not share amino acid sequence similarity with Fst1 and -b. Both fstl1a and -b mRNAs were widely expressed in adult tissues. During embryogenesis, grass carp fstl1a and -b mRNA was detected in the presomitic mesoderm and somites at 12h post fertilization (hpf). At 24hpf, fstl1a mRNA was expressed in the hindbrain, somites, notochord and tailbud, while fstl1b mRNA was only detected in the tailbud. At 36hpf, fstl1a mRNA was detected in the hindbrain and notochord, and fstl1b was also expressed in the notochord. Furthermore, fstl1a and -b were downregulated in brain and liver tissue following injection with 10 or 50µg hGH, while fstl1b was significantly up-regulated in muscle tissue after 10µg hGH treatment. Both fstl1a and -b were significantly up-regulated at 2, 4 or 6days of nutrient restriction, and fstl1a was still highly expressed in the liver and muscle after 3days of refeeding, as was fstl1b in the brain and muscle. The expression of these genes returned to near control levels following 6days of refeeding. Our findings suggest that the two fstls play important but divergent roles in embryonic development and tissue growth regulation in grass carp.

Functional conservation and divergence of duplicated fibroblast growth factor receptor 1 (fgfr1) genes in blunt snout bream (Megalobrama amblycephala).

Fgfr1 is a fibroblast growth factor receptor involved in regulating cell growth, proliferation, differentiation and migration. Here, we report the isolation and characterization of duplicated fgfr1 genes in blunt snout bream (Megalobrama amblycephala). Blunt snout bream fgfr1a and -1b cDNAs were found to share a relatively high sequence identity of 82%. During embryogenesis, both fgfr1a and -1b mRNAs were highly detected at zygotes but gradually decreased and then constantly expressed after 16hpf, besides a strong expression for the fgfr1b mRNA at 12hpf. Whole-mount in situ hybridization demonstrated that fgfr1a mRNA was transcribed at the eyes, mid-hindbrain boundary (MHB), brain, posterior somites and tailbud at 16hpf, while the fgfr1b mRNA was only detected at the eyes and posterior somites at the same period. At 28hpf embryos, both fgfr1a and -1b mRNAs were expressed in the eyes, brain, pharyngeal arches and tailbud, and in the eyes, brain, pharyngeal arches and notochord at 55hpf. In adult fish, fgfr1a mRNA was strongly expressed in the gill, gonad, brain and midgut, but examined relatively low in the skin and kidney. In contrast, the fgfr1b mRNA was highly detected in the brain and liver and quite low in the skin, gill and kidney. During starvation, both fgfr1a and -1b mRNAs were significantly up-regulated in the intestine and liver, but down-regulated in the brain. Moreover, duplicated fgfr1 mRNAs were differentially inhibited in tissues with exogenous recombinant hGH. Our results suggest that two fgfr1 genes play important roles in regulating growth and development in blunt snout bream.

In vitro induction of human adipose-derived stem cells into lymphatic endothelial-like cells.

Human adipose-derived stem cells (hADSCs) may provide a suitable number of progenitors for the treatment of lymphatic edema; however, to date the protocols for inducing hADSCs into this tissue type have not been standardized. We wished to investigate the induction of hADSCs into lymphatic endothelial-like cells using vascular endothelial growth factor-C156S (VEGF-C156S) and other growth factors in vitro. hADSCs from healthy adult adipose tissue were purified using enzyme digestion. Differentiation was induced using medium containing VEGF-C156S and bovine fibroblast growth factor (bFGF). Differentiation was confirmed using immunostaining for lymphatic vessel endothelial hyaluronan receptor (LYVE-1) and fms-related tyrosine kinase 4 (FLT-4), two lymphatic endothelial cell markers. The expression levels of LYVE-1, prospero homeobox 1 (PROX-1), and FLT-4 throughout induction were assessed using reverse transcriptase quantitative polymerase chain reaction. hADSCs were successfully obtained by trypsin digest and purification. Flow cytometry showed these cells were similar to mesenchymal stem cells, with a high positive rate of CD13, CD29, CD44, and CD105, and a low positive rate of CD31, CD34, CD45, and HLA-DR. Induction to lymphatic endothelial-like cells was successful, with cells expressing high levels of LYVE-1, PROX-1, and FLT-4. Adipose-derived stem cells can be induced to differentiate into lymphatic endothelial-like cells using a medium containing VEGF-C156S, bFGF, and other growth factors. This population of lymphatic endothelial-like cells may be useful for lymphatic reconstruction in the future.

Duplicated connective tissue growth factor genes in hypoxia-sensitive blunt snout bream Megalobrama amblycephala and their in vivo expression.

Connective tissue growth factor (CTGF) is a peptide involved in tissue growth and development, and can be regulated by hypoxia stress. This study aimed to isolate and characterize duplicate Ctgf genes in blunt snout bream Megalobrama amblycephala, and determine their expression patterns and response to hypoxia. The blunt snout bream Ctgfa and Ctgfb were found to be highly divergent, sharing a relatively low sequence identity of 57%. During embryogenesis, Ctgfa mRNA expression levels were low, gradually decreased from zygotes to 12h post-fertilization (hpf), markedly increased from 16 hpf, and then stabilized from 32 to 40 hpf. Ctgfb expression levels were constant but low from zygotes to 20 hpf, then gradually increased from 24 to 40 hpf. Ctgfa mRNA was expressed in the adaxial cells of the somites, floor plate, and tailbud at 24 hpf, and in the notochord and ethmoid plate at 36 hpf, whereas Ctgfb mRNA was weakly expressed in the adaxial cells and floor plate at 24 hpf, and in the notochord at 36 hpf. In adult fish, Ctgfa mRNA was strongly expressed in the kidney, brain, intestine, muscles, and skin, while Ctgfb mRNA was detected in all examined tissues. During hypoxic treatment, the mRNA levels of both Ctgfa and -b were significantly upregulated in the gill and liver, whereas Ctgfa mRNAs in the brain and kidney and Ctgfb mRNAs in the kidney significantly decreased. These results provide new insights into the functional conservation and divergence of Ctgf genes and reveal their responses to hypoxia.

PPARγ suppression inhibits adipogenesis but does not promote osteogenesis of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are the common progenitors of osteoblasts and adipocytes. A reciprocal relationship exists between osteogenesis and adipogenesis in the bone marrow, and the identification of signaling pathways that stimulate MSC osteogenesis at the expense of adipogenesis is of great importance from the viewpoint of developing new therapeutic treatments for bone loss. The adipogenic transcription factor peroxisome proliferator-activated receptor γ (PPARγ) has been reported to play a vital role in modulating mesenchymal lineage allocation within the bone marrow compartment, stimulating adipocyte development at the expense of osteoblast differentiation. Hence, PPARγ may be a valuable target for drugs intended to enhance bone mass. However, little direct evidence is available for the role played by PPARγ in human mesenchymal lineage allocation. In this study, using human MSCs as an in vitro model, we showed that the two isoforms of PPARγ, PPARγ1 and PPARγ2, were differentially induced during hMSC adipogenesis, whereas only PPARγ1 was detected during osteogenesis. BADGE and GW9662, two potential antagonists of PPARγ, as well as lentivirus-mediated knockdown of PPARγ, inhibited hMSC adipogenesis but did not significantly affect osteogenesis. PPARγ knockdown did not significantly influence the expression level of the osteogenic transcription factor Runx2. Together, these results suggest that PPARγ is not the master factor regulating mesenchymal lineage determination in human bone marrow.

Microproteinuria for detecting calcineurin inhibitor-related nephrotoxicity after liver transplantation.

AIM: To investigate whether microproteinuria could be used as an early and sensitive indicator to detect calcineurin inhibitor (CNI)-related nephrotoxicity after liver transplantation. METHODS: All liver transplant recipients with normal serum creatinine (SCr) and detectable microproteinuria at baseline were included in this study. The renal function was monitored by the blood clearance of (99m)Tc-diethylenetriaminepentaacetic acid every 6 mo. Microproteinuria, SCr and blood urea nitrogen (BUN) were measured at entry and at subsequent follow-up visits. The patients were divided into different groups according to the mean values of glomerular filtration rate (GFR) at the follow-up time points: Group 1, GFR decreased from baseline by 0%-10%; Group 2, GFR decreased from baseline by 11%-20%; Group 3, GFR decreased from baseline by 21%-40%; Group 4, GFR decreased from baseline by > 40% and/or SCr was increasing. RESULTS: A total of 143 patients were enrolled into this study (23 females and 120 males). The mean follow-up was 32 mo (range 16-36 mo). Downward trends in renal function over time were observed in the study groups. SCr and BUN increased significantly only in Group 4 patients (P < 0.001). beta2-microglobulin (beta2m) and alpha1-microglobulin (alpha1m) significantly increased with the subtle change of renal function in recipients who were exposed to CNI-based immunosuppression regimens. The reductions in GFR were closely correlated with elevated alpha1m (r(2) = -0.728, P < 0.001) and beta2m (r(2) = -0.787, P < 0.001). CONCLUSION: beta2m and alpha1m could be useful as early and sensitive indicators of CNI-induced nephrotoxicity.

[Estimation formula of standard liver volume for Chinese adults].

UNLABELLED: We retrospectively reviewed 115 cases of living donors for right lobes of liver transplants, not including the middle hepatic veins, in our hospital. The weights and volumes of the right lobe liver grafts were measured and recorded in the surgery. The actual total volume of liver was calculated from the right lobe graft volume divided by the proportion of the right lobe as indicated in the computed tomography. Simple linear regression analysis and stepwise multiple linear regression analysis were undertaken to develop a formula for calculating the total liver volume, which was compared with other existing formulae. RESULTS: The liver donors had a mean age of (35.97 +/- 9.6) years old, and a female to male ratio of 60:55. The computed tomography detected a mean volume of (727.47 +/- 136.17) mL of right lobe, which occupied 55.59% +/- 6.70% of the entire livers. The right lobe grafts had an actual volume of (581.73 +/- 96.137) mL and the donors had an actual total liver volume of (1053.08 +/- 167.56) mL. A formula was developed for calculating the standard liver volume: SLV(mL) = 11.508 x Body Weight + 334.024. The HongKong formula underestimated, whereas all of the other formulae overestimated the actual liver volume of the donors. Conclusion The standard liver volume of Chinese adults can be calculated as: SLV = 11.508 x Body Weight + 334.024.

[Evaluation of living donor liver transplantation for patients with hepatocellular carcinoma].

OBJECTIVE: To evaluate the donor risks and potential recipient benefits of living donor liver transplantation (LDLT) for adult patients with hepatocellular carcinoma (HCC). METHODS: From January 2002 to December 2006, a total of 27 LDLT for HCC patients were performed in our center, of which 25 received right lobe grafts and 2 received dual grafts. The clinical and follow-up data of these 27 recipients and 29 donors were analyzed retrospectively. RESULTS: Of the 29 donors, the overall complication rate was 17.24% (5 cases). Two cases (6.90%) experienced major complications (one with intra-abdominal bleeding and one with portal vein thrombosis) and three cases (10.34%) experienced minor ones (fat necrosis and infection of the surgical skin wound in one, pleural effusion in another and transient chyle leakage in the third). All donors were fully recovered and returned to their previous work. No recipients developed small-for-size syndrome. The overall HCC patients survival rate at 1- and 3-years was 84.01% and 71.40%, respectively, similar to that of patients undergoing LDLT for various nonmalignant diseases during the same period (P > 0.05). CONCLUSION: Although further study is needed to fully assess the risks and benefits of LDLT for the HCC patients and donors, our present results preliminarily suggest that LDLT offers an acceptable chance and duration of survival in patients with HCC, and it is a relatively safe procedure.

Donor safety in adult living donor liver transplantation using the right lobe: single center experience in China.

AIM: To evaluate the safety of donors in adult living donor liver transplantation (LDLT) using the right lobe in a single liver transplantation center in China. METHODS: We investigated retrospectively 52 living donor liver resections performed from October 2003 to July 2006. All patients were evaluated by blood tests and abdominal CT. The mean donor age was 28.2 +/- 7.4 years. Residual liver volume was 42.1% +/- 4.7%. Mean operative time was 420 +/- 76.2 min; mean ICU stay, less than 36 h; mean hospital stay, 16.4 +/- 8.6 d; and mean follow-up period, 6 mo. RESULTS: There was no mortality. The overall complication rate was 40% (21 donors). Major complications included biliary leak in two, and pneumonia in 2 donors. Minor complications included mild pleural effusion in 12 donors, transient ascites in 6, mild depression in 4, intra-abdominal collections in 2, and wound infections in 1 donor. Residual liver volume did not affect the complication rate. None required re-operation. Return to pre-donation activity occurred within 5-8 wk. CONCLUSION: Right hemi-hepatectomy can be performed safely with minimal risk in cases of careful donor selection. Major complications occurred in only 7.7% of our series.