Biomedical subjects

Li-Jun Yang

Publications and source records attributed to Li-Jun Yang.

10 recordsLinked to original sources

PubMed2008

Pediatric T-cell post-transplant lymphoproliferative disorder after solid organ transplantation.

Post-transplant lymphoproliferative disorder (PTLD) is the most common treatment related malignancy that occurs after solid organ transplantation (SOT). PTLD has extended from its initial description as an Epstein-Barr virus (EBV)-driven B-cell proliferation to include EBV-negative and non B-lineage cases. T-cell PTLD (T-PTLD) is rare in both adults and children. We report two cases of pediatric T-PTLD after SOT (liver and lungs) and review cases reported in the literature. Both patients had a bimodal response to therapy with initial eradication of bulky nodal disease with regimens typically used to treat leukemia, but persistence of low-level clonal T-cells in marrow, CSF and lung in one case.

Child, Preschool↗

PubMed2006

Role of Pax4 in Pdx1-VP16-mediated liver-to-endocrine pancreas transdifferentiation.

Although Pdx1-VP16 expression induces hepatic cell transdifferentiation into pancreatic precursor cells (WB-1), these incompletely reprogrammed cells fail to become glucose-sensitive insulin-producing cells in the absence of the activation of late-stage pancreatic transcription factors. As Pax4 promotes late-stage beta-cell differentiation and maturation, we generated lentiviral vector (LV) containing mouse Pax4 gene and developed two hepatic cell lines expressing Pax4 in the absence (WB-2 cells) or presence (WB-1A cells) of Pdx1-VP16, via LV-mediated gene transfer. Functional Pax4 protein expression in WB-2 and WB-1A cells was confirmed by electrophoretic mobility shift assay and Pdx1-VP16 protein expression in WB-1 and WB-1A cells was confirmed by Western blotting. Activation of Pax4 resulted in the expression of the late-stage transcription factors, including Pax6, Isl-1, and MafA, and generated a gene expression profile for WB-1A cells similar to that of functional rat insulinoma INS-1 cells. Insulin abundance in WB-1A cells was demonstrated by immunostaining. WB-1A cells exhibited glucose-responsive insulin release in vitro, and caused a rapid reversal of hyperglycemia following cell transplantation into streptozotocin-induced diabetic mice. Intraperitoneal glucose tolerance test showed a normal glucose response in WB-1, and WB-1A transplanted mice similar to that of normal mice. Removal of transplanted WB-1A cells resulted in a return of hyperglycemia, confirming that they were responsible for the observed normoglycemia. The explanted WB-1A cells exhibited strong insulin staining comparable to native islet beta-cells. These studies indicate that activation of Pax4 in Pdx1-VP16-expressing cells reprograms pancreatic precursor-like WB-1 cells into glucose-responsive, more mature insulin-producing cells.

Animals↗

PubMed2006

Reprogramming liver-stem WB cells into functional insulin-producing cells by persistent expression of Pdx1- and Pdx1-VP16 mediated by lentiviral vectors.

Adenovirus-mediated transient expression of the pancreatic duodenal homeobox transcription factor Pdx1 in mouse liver activates pancreatic endocrine and exocrine genes, the latter reportedly resulting in severe hepatitis. Expression of a super-active form of Pdx1 or Pdx1-VP16 selectively transdifferentiates hepatic WB cells into functional pancreatic beta-like insulin-producing cells, without evidence of exocrine differentiation. No study has systematically compared the transdifferentiation efficiency of Pdx1 and Pdx1-VP16 at the cellular and molecular level. Comparisons can be ambiguous when vectors harboring a transcription factor cDNA have differing extents and duration of gene expression. In view of the remarkable capacity of lentiviral vector (LV) for delivering and integrating transgene into both dividing and nondividing cells, we transduced rat hepatic stem cell-like WB cells with LV-Pdx1 or LV-Pdx1-VP16, and then used the limiting-dilution technique to clone single-cell-derived cell lines that stably express either Pdx1 or Pdx1-VP16. With these cell lines, we studied: (a) the expression of Pdx1 or Pdx1-VP16 protein by Western blotting and immunocytochemistry; (b) the repertoire of long-term expression of Pdx1- or Pdx1-VP16-induced pancreatic gene expression using RT-PCR methods; and (c) their capacity to serve as beta-cell surrogates in restoring euglycemia in streptozotocin-treated diabetic mice. We found that cell lines expressing either Pdx1 or Pdx1-VP16 long-term exhibited similar profiles for expression of genes related to pancreatic development and beta-cell function, and reversed hyperglycemia in diabetic mice. We also examined short-term expression of Pdx1 or Pdx1-VP16, and the results demonstrated that expression of Pdx1-VP16 is more efficient in initiating liver-to-endocrine pancreas transdifferentiation. Our findings demonstrate: (a) that the LV system is highly effective in producing persistent expression of Pdx1 or Pdx1-VP16 in WB hepatic cells; and (b) long-term, persistent expression of either Pdx1 or Pdx1-VP16 is similarly effective in converting hepatic stem cells into pancreatic endocrine precursor cells that, upon transplantation into diabetic mice, become functional insulin-producing cells and restore euglycemia.

Animals↗

PubMed2005

Liver stem cell-derived beta-cell surrogates for treatment of type 1 diabetes.

Consistent with the common embryonic origin of liver and pancreas as well the similar glucose-sensing systems in hepatocytes and pancreatic beta-cells, it should not be surprising that liver stem cells/hepatocytes can transdifferentiate into insulin-producing cells under high-glucose culture conditions or by genetic reprogramming. Persistent expression of the pancreatic duodenal homeobox-1 (Pdx1) transcription factor or its super-active form Pdx1-VP16 fusion protein in hepatic cells reprograms these cells into pancreatic beta-cell precursors. In vitro culture at elevated glucose concentrations or in vivo exposure to a hyperglycemia are required for further differentiation and maturation of liver-derived pancreatic beta-cell precursor into functional insulin-producing pancreatic beta-like cells. Under appropriate conditions, multiple pancreatic transcription factors can work in concert to reprogram liver stem/adult liver cells into functional insulin-producing cells. If such autologous liver-derived insulin-producing cells can be made to escape the type 1 diabetes-associated autoimmunity, they may serve as a valuable cell source for future cell replacement therapy without the need for life-long immunosuppression.

Animals↗

PubMed2005

Genetic engineering of human embryonic stem cells with lentiviral vectors.

Human embryonic stem (hES) cells present a valuable source of cells with a vast therapeutic potential. However, the low efficiency of directed differentiation of hES cells remains a major obstacle in their uses for regenerative medicine. While differentiation may be controlled by the genetic manipulation, effective and efficient gene transfer into hES cells has been an elusive goal. Here, we show stable and efficient genetic manipulations of hES cells using lentiviral vectors. This method resulted in the establishment of stable gene expression without loss of pluripotency in hES cells. In addition, lentiviral vectors were effective in conveying the expression of an U6 promoter-driven small interfering RNA (siRNA), which was effective in silencing its specific target. Taken together, our results suggest that lentiviral gene delivery holds great promise for hES cell research and application.

Cell Differentiation↗

PubMed2005

Derivation of adipocytes from human embryonic stem cells.

Human embryonic stem (hES) cells are undifferentiated and pluripotent cells that hold great therapeutic potential, but are hampered by our limited knowledge to promote specific cell differentiation. Here we provide the first report of the directed differentiation of hES cells into adipocytes. Embryoid bodies (EBs) derived from hES cells are shown to respond to factors that promote adipogenesis. Differentiated cells were observed that displayed the key features of adipocytes, i.e., expression of specific molecular markers, such as peroxisome proliferator-activated receptor gamma2 (PPARgamma2), adipocyte fatty acid binding protein (aP2) and adiponectin, the secretion of leptin, and the accumulation of lipid droplets in cytoplasm. Taken together, our results demonstrate that adipocytes derived from hES cells in vitro can provide a novel model system to study human adipogenesis and obesity.

Adipocytes↗

PubMed2004

[The expression profiles of human chromosome 21 orthologues in mouse M II oocyte].

OBJECTIVE: To analyse the expression profile of orthologues of human chromosome 21 (HC21) in mouse M II oocytes, and to discuss the relationship between this expression profile and early embryonic development and further to find the possible reasons of DS phenotypes genesis. METHODS: cDNA array and Global RT-PCR methods were used to analyse and identify the expression profile of 93 HC21 orthologues in mouse M II oocytes. RESULTS: 26 of 93 orthologues were proved to be expressed in mouse M II oocytes and these genes were involved in many biological procedure including transcriptional, metabolism, ionic channel, and ubiquitin pathway etc. CONCLUSION: To our knowledge, this is first report about the HC21 orthologues expression profile in M II oocytes and this expression profile indicates that 26 of 93 HC21 orthologues may directly play important role in early development, and the biological information obtained from this experiment would be beneficial to understand imbalance of HC21 genes expression and the molecular mechanism of DS phenotypes genesis.

Animals↗

PubMed2004

High glucose is necessary for complete maturation of Pdx1-VP16-expressing hepatic cells into functional insulin-producing cells.

Pdx1 has been shown to convert hepatocytes into both exocrine and endocrine pancreatic cells in mice, but it fails to selectively convert hepatocytes into pure insulin-producing cells (IPCs). The molecular mechanisms underlying the transdifferentiation remain unclear. In this study, we generated a stably transfected rat hepatic cell line named WB-1 that expresses an active form of Pdx1 along with a reporter gene, RIP-eGFP. Our results demonstrate that Pdx1 induces the expression of multiple genes related to endocrine pancreas development and islet function in these liver cells. We do not however find any expression of the late-stage genes (Pax4, Pax6, Isl-1, and MafA) related to beta-cell development, and the cells do not secrete insulin upon the glucose challenge. Yet when WB-1 cells are transplanted into diabetic NOD-scid mice, these genes become activated and hyperglycemia is completely reversed. Detailed comparison of gene expression profiles between pre- and posttransplanted WB-1 cells demonstrates that the WB-1 cells have similar properties as that seen in pancreatic beta-cells. In addition, in vitro culture in high-glucose medium is sufficient to induce complete maturation of WB-1 cells into functional IPCs. In summary, we find that Pdx1-VP16 is able to selectively convert hepatic cells into pancreatic endocrine precursor cells. However, complete transdifferentiation into functional IPCs requires additional external factors, including high glucose or hyperglycemia. Thus, transdifferentiation of hepatocytes into functional IPCs may serve as a viable therapeutic option for patients with type 1 diabetes.

Animals↗

PubMed2004

In vivo and in vitro characterization of insulin-producing cells obtained from murine bone marrow.

Efforts toward routine islet cell transplantation as a means for reversing type 1 diabetes have been hampered by islet availability as well as allograft rejection. In vitro transdifferentiation of mouse bone marrow (BM)-derived stem (mBMDS) cells into insulin-producing cells could provide an abundant source of autologous cells for this procedure. For this study, we isolated and characterized single cell-derived stem cell lines obtained from mouse BM. In vitro differentiation of these mBMDS cells resulted in populations meeting a number of criteria set forth to define functional insulin-producing cells. Specifically, the mBMDS cells expressed multiple genes related to pancreatic beta-cell development and function (insulin I and II, Glut2, glucose kinase, islet amyloid polypeptide, nestin, pancreatic duodenal homeobox-1 [PDX-1], and Pax6). Insulin and C-peptide production was identified by immunocytochemistry and confirmed by electron microscopy. In vitro studies involving glucose stimulation identified glucose-stimulated insulin release. Finally, these mBMDS cells transplanted into streptozotocin-induced diabetic mice imparted reversal of hyperglycemia and improved metabolic profiles in response to intraperitoneal glucose tolerance testing. These results indicate that mouse BM harbors cells capable of in vitro transdifferentiating into functional insulin-producing cells and support efforts to derive such cells in humans as a means to alleviate limitations surrounding islet cell transplantation.

Animals↗

PubMed2003

[Gene cloning and prokaryotic expression of the PID domain of human DOC-2 molecule].

AIM: To clone the PID domain of human DOC-2 (nDOC-2) and express it in E.coli DH5alpha. METHODS: The cDNA fragment encoding the PID domain of nDOC-2 was amplified by RT-PCR from normal human ovarian tissue and cloned to pUC19. The DNA fragment from the pUC19-nDOC-2 digested with BamH I and EcoR I was ligated to the BamH I/EcoR I digested prokaryotic expression vector pGEX-4T-1.The expression of fusion protein was induced with IPTG and the expressed product was identified by SDS-PAGE. RESULTS: (1)The sequencing and endonucleases digestion analysis showed that the fragment of nDOC-2 gene was insert into vectors pUC19 and pGEX-4T-1;(2)SDS-PAGE showed the nDOC-2 gene had been expressed in E.coli DH5alpha. CONCLUSION: The PID domain of nDOC-2 was expressed successfully in prokaryote, which makes preparation for further researching the function of DOC-2 and preparing antibodies to DOC-2 protein.

Cloning, Molecular↗