Ludi Zhang

Direct Reprogramming of Human Fibroblasts to Functional and Expandable Hepatocytes

The generation of large numbers of functional human hepatocytes for cell-based approaches to liver disease is an important and unmet goal. Direct reprogramming of fibroblasts to hepatic lineages could offer a solution to this problem but so far has only been achieved with mouse cells. Here, we generated human induced hepatocytes (hiHeps) from fibroblasts by lentiviral expression of FOXA3, HNF1A, and HNF4A. hiHeps express hepatic gene programs, can be expanded inxa0vitro, and display functions characteristic of mature hepatocytes, including cytochrome P450 enzyme activity and biliary drug clearance. Upon transplantation into mice with concanavalin-A-induced acute liver failure and fatal metabolic liver disease due to fumarylacetoacetate dehydrolase (Fah) deficiency, hiHeps restore the liver function and prolong survival. Collectively, our results demonstrate successful lineage conversion of nonhepatic human cells into mature hepatocytes with potential for biomedical and pharmaceutical applications.

Improved survival of porcine acute liver failure by a bioartificial liver device implanted with induced human functional hepatocytes.

Acute liver failure (ALF) is a life-threatening illness. The extracorporeal cell-based bioartificial liver (BAL) system could bridge liver transplantation and facilitate liver regeneration for ALF patients by providing metabolic detoxification and synthetic functions. Previous BAL systems, based on hepatoma cells and non-human hepatocytes, achieved limited clinical advances, largely due to poor hepatic functions, cumbersome preparation or safety concerns of these cells. We previously generated human functional hepatocytes by lineage conversion (hiHeps). Here, by improving functional maturity of hiHeps and producing hiHeps at clinical scales (3 billion cells), we developed a hiHep-based BAL system (hiHep-BAL). In a porcine ALF model, hiHep-BAL treatment restored liver functions, corrected blood levels of ammonia and bilirubin, and prolonged survival. Importantly, human albumin and α-1-antitrypsin were detectable in hiHep-BAL-treated ALF pigs. Moreover, hiHep-BAL treatment led to attenuated liver damage, resolved inflammation and enhanced liver regeneration. Our findings indicate a promising clinical application of the hiHep-BAL system.

The ultraviolet laser from individual ZnO microwire with quadrate cross section

The ZnO microwires with quadrate cross section were synthesized by chemical vapor deposition method. The ultraviolet laser with the Fabry-pérot cavity modes was realized from an individual ZnO microwire. Under the low excitation power densities, the amplified spontaneous emission was observed from the ZnO microwire, while the lasing action was observed under the high excitation power densities. The ZnO microwire exhibited low threshold excitation intensity of 58 kW/cm(2) and quality factor of 485. The characteristics and possible lasing mechanism were investigated in detail.

Cell fate conversion: Direct induction of hepatocyte-like cells from fibroblasts

One of the essential features of stem cells is their cellular plasticity to differentiate into daughter cells with defined functions. Recently, induction of pluripotent stem cells from somatic cells by defined transcription factors led to the focus on cellular plasticity of terminally differentiated cells. This approach is adopted by other studies to demonstrate the cell fate conversion between different lineages of terminally differentiated cells. We and others showed that induced hepatocyte‐like (iHep) cells are directly converted from mouse fibroblasts by overexpression of liver‐enriched transcription factors. iHep cells as well as pluripotent stem cell‐ or mesenchymal stem cell‐derived hepatocyte‐like cells provide potential cell sources for disease modeling, transplantation, and tissue engineering independent of donor organs. Here, we review the latest advances in generating hepatocyte‐like cells and summarize general criteria for evaluating these cells. In addition, we propose a possible role of the p19Arf/p53 pathway in cell fate maintenance, which apparently limits the formation of induced pluripotent stem (iPS) cells and iHep cells. J. Cell. Biochem. 114: 256–265, 2013.

Directional transport of fast electrons at the front target surface irradiated by intense femtosecond laser pulses with preformed plasma

The effects of laser incidence angle on lateral fast electron transport at front target surface, when a plasma is preformed, irradiated by intense (>10(18) W/cm(2)) laser pulses, are studied by K-alpha imaging technique and electron spectrometer. A horizontally asymmetric K-alpha halo, resulting from directional lateral electron transport and energy deposition, is observed for a large incidence angle (70 degrees). Moreover, a group of MeV high energy electrons is emitted along target surface. It is believed that the deformed preplasma and the asymmetrical distribution of self-generated magnetic field, at large incidence angle, play an important role in the directional lateral electron transport.

Efficient liver repopulation of transplanted hepatocyte prevents cirrhosis in a rat model of hereditary tyrosinemia type I

Hereditary tyrosinemia type I (HT1) is caused by a deficiency in the enzyme fumarylacetoacetate hydrolase (Fah). Fah-deficient mice and pigs are phenotypically analogous to human HT1, but do not recapitulate all the chronic features of the human disorder, especially liver fibrosis and cirrhosis. Rats as an important model organism for biomedical research have many advantages over other animal models. Genome engineering in rats is limited till the availability of new gene editing technologies. Using the recently developed CRISPR/Cas9 technique, we generated Fah−/− rats. The Fah−/− rats faithfully represented major phenotypic and biochemical manifestations of human HT1, including hypertyrosinemia, liver failure, and renal tubular damage. More importantly, the Fah−/− rats developed remarkable liver fibrosis and cirrhosis, which have not been observed in Fah mutant mice or pigs. Transplantation of wild-type hepatocytes rescued the Fah−/− rats from impending death. Moreover, the highly efficient repopulation of hepatocytes in Fah−/− livers prevented the progression of liver fibrosis to cirrhosis and in turn restored liver architecture. These results indicate that Fah−/− rats may be used as an animal model of HT1 with liver cirrhosis. Furthermore, Fah−/− rats may be used as a tool in studying hepatocyte transplantation and a bioreactor for the expansion of hepatocytes.

X-ray lasers from Inner-shell transitions pumped by the Free-electron laser

We present a approach of generating femtosecond coherent x-ray pulses by using the self-amplified free-electron laser (SASE FEL) to pump the inner-shell x-ray lasers (ISXRLs). Theoretical simulations are performed. The gain characteristics are analyzed for the two representative schemes of inner-shell x-ray transitions, ie. the self-terminated x-ray lasing (1s)(-1)-->(2p)(-1) (lambda = 4.5nm) in carbon (Z = 6) and the quasi-stationary x-ray lasing (2p)(-1 )-->(3s)(-1) (lambda = 4.1nm) in calcium (Z = 20). When the 10fs x-ray FEL pulses are available at 284eV and 360eV with the pumping intensities of 1.2x10(15)W/cm(2) and 2x10(17)W/cm(2) for C and Ca, respectively, a net gain of 140cm(-1) can be predicted. Using a one-dimensional model, the properties of output ISXRLs are studied. By the Carbon ISXRL scheme, the multi-spiky SASE FEL x-ray pulse with chaotic temporal structure is smoothed to a temporally continuous x-ray pulse with a comparable duration but at a different wavelength. The Calcium scheme, can be used to create one single x-ray laser pulse with a duration as short as 2fs. The spectral bandwidth of the output ISXRLs is an order of 10 (-3), which is one order narrower than that of the SASE FELs.

Distribution and dynamics of risk factors associated with highly pathogenic avian influenza H5N1

Within Chinas Poyang Lake region, close interactions between wild migratory birds and domestic poultry are common and provide an opportunity for the transmission and subsequent outbreaks of highly pathogenic avian influenza (HPAI) virus. We overlaid a series of ecological factors associated with HPAI to map the risk of HPAI in relation to natural and anthropogenic variables, and we identified two hotspots for potential HPAI outbreaks in the Poyang Lake region as well as three corridors connecting the two hotspot areas. In hotspot I, there is potential for migratory birds to bring new avian influenza (AI) strains that can reassort with existing strains to form new AI viruses. Hotspot II features high-density poultry production where outbreaks of endemic AI viruses are likely. The three communication corridors that link the two hotspots further promote HPAI H5N1 transmission and outbreaks and lead to the persistence of AI viruses in the Poyang Lake region. We speculate that the regions unevenly distributed poultry supply-and-demand system might be a key factor inducing HPAI H5N1 transmission and outbreaks in the Poyang Lake region.

Distinct Gene Expression and Epigenetic Signatures in Hepatocyte-like Cells Produced by Different Strategies from the Same Donor

Summary Hepatocyte-like cells (HLCs) can be generated through directed differentiation or transdifferentiation. Employing two strategies, we generated induced pluripotent stem cell (iPSC)-HLCs and hiHeps from the same donor cell line. Both types of HLCs clustered distinctly from each other during gene expression profiling. In particular, differences existed in gene expression for phase II drug metabolism and lipid accumulation, underpinned by H3K27 acetylation status in iPSC-HLCs and hiHeps. While distinct phenotypes were achieved in vitro, both types of HLCs demonstrated similar phenotypes following transplantation into Fah-deficient mice. In conclusion, functional HLCs can be obtained from the same donor using two strategies. Global gene expression defined the differences between those populations in vitro. Importantly, both HLCs displayed partial but markedly improved hepatic function following transplantation in vivo, demonstrating plasticity and the potential for cell-based modeling in the dish and cell-based therapy in the future.

Cas9-nickase–mediated genome editing corrects hereditary tyrosinemia in rats

Hereditary tyrosinemia type I (HTI) is a metabolic genetic disorder caused by mutation of fumarylacetoacetate hydrolase (FAH). Because of the accumulation of toxic metabolites, HTI causes severe liver cirrhosis, liver failure, and even hepatocellular carcinoma. HTI is an ideal model for gene therapy, and several strategies have been shown to ameliorate HTI symptoms in animal models. Although CRISPR/Cas9-mediated genome editing is able to correct the Fah mutation in mouse models, WT Cas9 induces numerous undesired mutations that have raised safety concerns for clinical applications. To develop a new method for gene correction with high fidelity, we generated a Fah mutant rat model to investigate whether Cas9 nickase (Cas9n)-mediated genome editing can efficiently correct the Fah. First, we confirmed that Cas9n rarely induces indels in both on-target and off-target sites in cell lines. Using WT Cas9 as a positive control, we delivered Cas9n and the repair donor template/single guide (sg)RNA through adenoviral vectors into HTI rats. Analyses of the initial genome editing efficiency indicated that only WT Cas9 but not Cas9n causes indels at the on-target site in the liver tissue. After receiving either Cas9n or WT Cas9-mediated gene correction therapy, HTI rats gained weight steadily and survived. Fah-expressing hepatocytes occupied over 95% of the liver tissue 9 months after the treatment. Moreover, CRISPR/Cas9-mediated gene therapy prevented the progression of liver cirrhosis, a phenotype that could not be recapitulated in the HTI mouse model. These results strongly suggest that Cas9n-mediated genome editing is a valuable and safe gene therapy strategy for this genetic disease.