Transplanted human neural precursor cells integrate into the host neural circuit and ameliorate neurological deficits in a mouse model of traumatic brain injury.

Traumatic brain injury (TBI) is to date one of the major critical conditions causing death and disability worldwide. Exogenous neural stem/precursor cells (NSCs/NPCs) hold great promise for improving neurological dysfunction, but their functional properties in vivo remain unknown. Human neural precursor cells (hNPCs) carrying one fluorescent reporter gene (DsRed) can be observed directly in vivo using two-photon laser-scanning microscope. Therefore, we evaluated the neural integration and potential therapeutic effect of hNPCs on mice with TBI. Behavioral tests were performed by rotarod task and Morris Water Maze task. Neural integration was detected by fluorometric Ca2+ imaging and nerve tracing. We found that motor and cognition functions were significantly improved in mice with hNPCs injection compared to mice with vehicle treatment, and hNPCs integrated into the host circuit and differentiated toward neuronal lineage. Our study provided reliable evidence for further hNPCs transplantation in clinical practice.

[Differentiation of in vitro induced human pluripotent stem cells into hematopoietic stem/progenitor cells].

This study was aimed to explore the differentiation of in vitro induced human pluripotent stem cells (iPSC) into hematopoietic stem progenitor cells. The human iPSC were induced to differentiate into hematopoietic stem/progenitor cell by co-culturing with OP9 bone marrow stromal cells. The expression of hematopoietic stem/progenitor cell surface markers were detected by flow cytometry. The regulation gene expressions of iPSC and hematopoietic stem/progenitor cells were measured by real-time PCR. The CD34(+) hematopoietic stem/progenitor cells were isolated by using immunomagnetic beads, and were used for colony formation assay. The results showed that after iPSC were co-cultured with OP9 cells for 4 days, the morphological changes of iPSC could be observed. Hematopoietic stem/progenitor cell surface markers CD34 and CD43 could be detected by flow cytometry after differentiation. The pluripotent marker gene OCT4 expression gradually decreased and blood-related transcription factor Gata-2 expression gradually increased, while Runx-1 expression was wavily changed, CD34 expression gradually increased. The erythroid colony(CFU-E), granulocyte colony(CFU-G), megakaryocytic colony(CFU-M), granulocyte-megakaryocytic colony(CFU-GM), and mixed colony(CFU-GEMM) were obtained after cultures for 14 d. It is concluded that the human iPSC cells can be induced to differentiate into hematopoietic stem/progenitor cells in vitro by co-culture with OP9 cells.

Antibody-directed double suicide gene therapy targeting of MUC1- positive leukemia cells in vitro and in vivo.

Our aim was to specifically transfer the cytosine deaminase (CD) and thymidine kinase (TK) genes into mucin 1 (MUC1)-positive leukemia cells by anti-MUC1 antibody directed infection of replication-defective lentivirus and to evaluate the targeted cytotoxicity of double suicide genes to leukemia. The target gene vector (containing CD and TK) and envelope (containing GFP and anti-MUC1) and packaging plasmids were cotransfected into 293T cells to produce the recombinant lentivirus. Suicide genes in virus-infected leukemia cells (U937, Jurkat, and K562) were detected by western blot. The cytotoxicity and bystander effect in vitro and the therapeutic effect in vivo were detected after treatment with the prodrugs. The results revealed that combined treatment with prodrug 5-fluorocytosine (5-FC) and ganciclovir (GCV) inhibited leukemia cell growth and caused significant bystander effect than treatment with either prodrug alone. TK/GCV treatment alone induced degeneration and cell death while the effect of CD/5-FC alone mainly caused vacuolar degeneration and necrosis. The addictive effects of combinatorial use of GCV and 5-FC mainly induced swelling of the mitochondria followed by necrosis of the leukemia cells. In vivo experiments revealed that both single and combinatorial prodrug treatments could prolong the survival time of leukemic mice. In summary, anti-MUC1 antibody directed lentiviral vector successfully transduced dual suicide genes and exerted targeted cytotoxicity against MUC1 positive leukemia cells. This targeted lentiviral dual suicide gene delivering system provides a promising approach for clinical treatment of leukemia in future.

[Experimental study on HDV ribozyme in vitro cleaving the HBV derived RNA fragment].

OBJECTIVE: To explore the possibility of transacting hepatitis D virus (HDV) ribozyme cleaving in vitro the hepatitis B virus (HBV) mRNA fragments. METHODS: According to the established pseudoknot-like structure, its' H1 domain was changed to design the transacting HDV ribozyme Rc1 and Rc2, which targeted the 701-713 site and 776-788 site of HBV C domain. After the chemically synthesised cDNA of the ribozyme was cloned into the vector PGEM-4Z, the transacting HDV ribozyme was transcriped using in vitro transcription technology. The in vitro cleavage characteristics of the ribozyme were studied and the kinetic parameters (Kcat and Km) were determined by Eadie Hofstee plotting. RESULTS: Both the two ribozymes had the ability to cleave the substrate, the cleavage percentage at 37 degrees for 90 minutes were 50% and 51%. According to the Eadie Hofstee plot, the Km of the Rc1 and Rc2 were 0.61 micromol and 0.58 micromol, the Kcat were 0.64 x min(-1) and 0.60 x min(-1),respectively. CONCLUSIONS: The cleaving ability of trans-acting HDV ribozyme on non-HDV RNA fragment was tested. The results showed a new potential of the antisense antisense regent for HBV gene therapy.

Identification of two distinct transactivation domains in the pluripotency sustaining factor nanog.

Nanog is a newly identified homeodomain gene that functions to sustain the pluripotency of embryonic stem cells. However, the molecular mechanism through which nanog regulates stem cell pluripotency remains unknown. Mouse nanog encodes a polypeptide of 305 residues with a divergent homeodomain similar to those in the NK-2 family. The rest of nanog contains no apparent homology to any known proteins characterized so far. It is hypothesized that nanog encodes a transcription factor that regulates stem cell pluripotency by switching on or off target genes. To test this hypothesis, we constructed fusion proteins between nanog and DNA binding domains of the yeast transcription factor Gal4 and tested the transactivation potentials of these constructs. Our data demonstrate that both regions N- and C- terminal to the homeodomain have transcription activities. Despite the fact that it contains no apparent transactivation motifs, the C-terminal domain is about 7 times as active as the N-terminal one. This unique arrangement of dual transactivators may confer nanog the flexibility and specificity to regulate downstream genes critical for both pluripotency and differentiation of stem cells.

Stem cell pluripotency and transcription factor Oct4.

Mammalian cell totipotency is a subject that has fascinated scientists for generations. A long lasting question whether some of the somatic cells retains totipotency was answered by the cloning of Dolly at the end of the 20th century. The dawn of the 21st has brought forward great expectations in harnessing the power of totipotentcy in medicine. Through stem cell biology, it is possible to generate any parts of the human body by stem cell engineering. Considerable resources will be devoted to harness the untapped potentials of stem cells in the foreseeable future which may transform medicine as we know today. At the molecular level, totipotency has been linked to a singular transcription factor and its expression appears to define whether a cell should be totipotent. Named Oct4, it can activate or repress the expression of various genes. Curiously, very little is known about Oct4 beyond its ability to regulate gene expression. The mechanism by which Oct4 specifies totipotency remains entirely unresolved. In this review, we summarize the structure and function of Oct4 and address issues related to Oct4 function in maintaining totipotency or pluripotency of embryonic stem cells.