Biomedical subjects

Linzhao Cheng

Publications and source records attributed to Linzhao Cheng.

26 records · Page 2Linked to original sources

PubMed2003

Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture.

Prolonged propagation of human embryonic stem (hES) cells is currently achieved by coculture with primary mouse embryonic fibroblasts (MEFs) serving as feeder cells. Unlike mouse ES cells, adding growth factors such as leukemia inhibitory factor is insufficient to maintain undifferentiated hES cells without feeder cells. The presence of uncharacterized rodent cells or crude extracts imposes a risk to the clinical applications of hES cells. While others looked for a replacement of MEFs with human fetal cells, we attempted to use easily accessible postnatal human cells such as human marrow stromal cells (hMSCs). Culture-expanded hMSCs from multiple donors were used as feeder cells to support growth of the H1 hES cell line under a serum-free culture condition. Human ES cell colonies cultured on irradiated hMSCs amplified >100-fold during the 30-day continuous culture (in five passages). The longest continuous expansion of hES cells on hMSCs tested to date is 13 passages. The expanded hES cells displayed the unique morphology and molecular markers characteristic of undifferentiated hES cells as observed when they were cultured on MEFs. They expressed the transcription factor Oct-4, a membrane alkaline phosphatase, and the stage-specific embryonic antigen (SSEA)-4, but not the SSEA-1 marker. Expanded hES cells on hMSCs retained unique differentiation potentials in culture and a normal diploid karyotype. The well-studied hMSCs (and this animal cell- and serum-free system) may provide a clinically and ethically feasible method to expand hES cells for novel cell therapies. In addition, this system may help to identify cytokines and adhesion molecules that are required for the self-renewal of hES cells.

Adult↗

PubMed2002

Transduction of donor hematopoietic stem-progenitor cells with Fas ligand enhanced short-term engraftment in a murine model of allogeneic bone marrow transplantation.

Fas-mediated apoptosis is a major physiologic mechanism by which activated T cells are eliminated after antigen-stimulated clonal expansion generates a specific cellular immune response. Because activated T cells are the major effectors of allograft rejection, we hypothesized that genetically modifying allogeneic bone marrow (BM) cells prior to transplantation could provide some protection from host T-cell attack, thus enhancing donor cell engraftment in bone marrow transplantation (BMT). We undertook studies to determine the outcome of lentiviral vector-mediated transduction of Fas ligand (FasL) into lineage antigen-negative (lin(-)) mouse BM cells (lin(-) BMs), in an allogeneic BMT model. FasL-modified lin(-) BMs killed Fas-expressing T cells in vitro. Mice that received transplants of allogeneic FasL(+) lin(-) BMs had enhanced short-term engraftment, after nonmyeloablative conditioning, as compared to controls. We observed no major hepatic toxicity or hematopoietic or immune impairment in recipient mice at these time points. These results suggest potential therapeutic approaches by manipulating lymphohematopoietic stem-progenitor cells to express FasL or other immune-modulating genes in the context of BMT.

Animals↗

PubMed2002

Dissection of the c-Kit signaling pathway in mouse primordial germ cells by retroviral-mediated gene transfer.

Establishment of the mammalian germ line is a prerequisite for fertility of the adult animal but we know surprisingly little about the molecular mechanisms regulating germ-line development in mammals. Signaling from the c-Kit receptor tyrosine kinase is essential for primordial germ cell (PGC) growth both in vivo and in vitro. Many downstream effectors of the c-Kit signaling pathway have been identified in other cell types but how these molecules control PGC survival and proliferation are unknown. Determination of the c-Kit effectors acting in PGCs has been hampered by the lack of effective methods to easily manipulate gene expression in these cells. We overcame this problem by testing the efficacy of retroviral-mediated gene transfer for manipulating gene expression in mammalian germ cells. We found that PGCs can be successfully infected with a variety of types of retroviruses. We used this method to demonstrate an important role for the AKT kinase in regulating PGC growth. Such technology for manipulating gene expression in PGCs will allow many of the molecular mechanisms regulating germ cell growth, behavior, and differentiation to be comprehensively analyzed.

Animals↗

PubMed2002

TEL-AML1, expressed from t(12;21) in human acute lymphocytic leukemia, induces acute leukemia in mice.

TEL-AML1 is expressed from the t(12;21) chromosomal translocation inB-precursor acute lymphocytic leukemia (ALL). Creation of the TEL-AML1fusion disrupts one copy of the TEL and AML1 genes, and loss of TEL or AML1 is also associated with cases of acute leukemia without TEL-AML1. To determine whether TEL-AML1 can contribute to leukemogenesis, we transduced marrow from C57BL/6 mice with a retroviral vector expressing TEL-AML1 or with a control vector. Transduced cells were introduced into irradiated syngeneic recipients. Two of 9 TEL-AML1 mice developed ALL (one T-lineage ALL and one B-precursor ALL), whereas 0 of 20 control mice developed leukemia. The B-precursor ALL was retransplantable and expressed TEL-AML1. We similarly transduced marrow from C57BL/6 mice lacking the overlapping p16(INK4a)p19(ARF) genes and transplanted the cells into wild-type recipients. No control mice died, but six of eight TEL-AML1/p16p19 mice died with leukemia. Overall, these findings indicate that TEL-AML1 contributes to leukemogenesis and may cooperate with loss of p16(INK4a)p14(ARF) to transform lymphoid progenitors.

3T3 Cells↗

PubMed2002

Acceleration of G(1) cooperates with core binding factor beta-smooth muscle myosin heavy chain to induce acute leukemia in mice.

The genes encoding the AML1 (RUNX1) or CBFbeta subunits of core binding factor (CBF) are commonly altered by translocation or mutation in human leukemias. Because CBF oncoproteins slow G(1), we sought to determine whether mutations that accelerate G(1) potentiate their ability to induce transformation. Wild-type or p16(INK4a)p19(ARF) (-/-) marrow cells transduced with CBFbeta-smooth muscle myosin heavy chain (SMMHC) were transplanted into wild-type, syngeneic recipients. CBFbeta-SMMHC significantly increased the development of acute leukemias from marrow lacking the overlapping p16p19 genes, based on analysis of Kaplan-Meier event-time distributions. Wild-type marrow was also transduced with vectors expressing either E7 alone or both E7 and CBFbeta-SMMHC. Combining oncogenes again increased leukemia formation. Exposing mice transplanted with CBFbeta-SMMHC-transduced cells to a mutagen, ethylnitrosourea, markedly accelerated leukemogenesis compared to expressing CBFbeta-SMMHC with loss of p16p19, indicating the need for multiple "hits" for transformation. The INV/p16p19 and INV/E7 leukemias were lymphoid and were clonal and retransplantable. Overall, these findings indicate that CBF mutations cooperate with genetic alterations that accelerate G(1) to induce acute leukemia.

Acute Disease↗

PubMed2002

Targeting transgene expression to antigen-presenting cells derived from lentivirus-transduced engrafting human hematopoietic stem/progenitor cells.

Hematopoietic stem cells (HSCs) represent an important target for the treatment of various blood disorders. As the source of critical cells within the immune system, genetic modification of HSCs can also be used to modulate immune responses. The effectiveness of HSC-mediated gene therapy largely depends on efficient gene delivery into long-term repopulating progenitors and targeted transgene expression in an appropriate progeny of the transduced pluripotent HSCs. Self-inactivating (SIN) lentiviral vectors have been demonstrated to be capable of transducing mitotically inactive cells, including HSCs, and accommodating a nonviral promoter to control the transgene expression in transduced cells. In this study, we constructed 2 SIN lentiviral vectors, EF.GFP and DR.GFP, to express the green fluorescent protein (GFP) gene controlled solely by the promoter of either a housekeeping gene EF-1alpha or the human HLA-DRalpha gene, which is selectively expressed in antigen-presenting cells (APCs). We demonstrated that both vectors efficiently transduced human pluripotent CD34+ cells capable of engrafting nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. When the EF.GFP vector was used, constitutive high-level GFP expression was obtained in all the human HSC progeny detectable in NOD/SCID mice and in subsequent in vitro differentiation assays, indicating that engrafting human HSCs have been transduced. In contrast, the DR.GFP vector mediated transgene expression specifically in human HLA-DR+ cells and highly in differentiated dendritic cells (DCs), which are critical in regulating immunity. Furthermore, human DCs derived from transduced and engrafted human cells potently stimulated allogeneic T-cell proliferation. This study demonstrated successful targeting of transgene expression to APCs/DCs after stable gene transduction of pluripotent HSCs.

Animals↗

PubMedDate not supplied

Functional antigen-presenting leucocytes derived from human embryonic stem cells in vitro.

BACKGROUND: Differentiated cells derived from pluripotent human embryonic stem (hES) cells offer the opportunity for new transplantation therapies. However, hES cells and their differentiated progeny express highly polymorphic MHC molecules that serve as major graft rejection antigens to the immune system of allogeneic hosts. To achieve sustained engraftment of donor cells, strategies must be developed to overcome graft rejection without broadly suppressing host immunity. One approach entails induction of donor-specific immune tolerance by establishing chimeric engraftment in hosts with haemopoietic cells derived from an existing hES cell line. We aimed to develop methods to efficiently differentiate hES cells to haemopoietic cells, including immune-modulating leucocytes, a prerequisite of the tolerance induction strategies applying to hES cell-mediated transplantation. METHODS: We developed a method to generate a broad range of haemopoietic cells from hES-generated embryonic bodies in the absence of murine stromal feeder cells. Embryonic bodies were further cultured in the presence of haemopoietic cytokines. In addition to flow cytometric analyses of haemopoietic cell markers, we analysed the hES cell-derived haemopoietic cells by colony-forming assays (for erythroid and myeloid progenitor cells), cytochemical staining, and mixed leucocyte reactions to determine the functional capacity of the generated antigen-presenting cells. FINDINGS: 12 independent experiments were done. When selected growth factors were added, leucocytes expressing CD45 were generated and released into culture media for 6-7 weeks. Under the condition used, both erythroid and myeloid progenitor cells were generated. About 25% of the generated leucocytes acquired MHC class II and costimulatory molecule expression. These hES-derived, MHC class II+ leucocytes resembled dendritic cells and macrophages, and they functioned as antigen-presenting cells capable of eliciting allogeneic CD4 and CD8 T-cell responses in culture. INTERPRETATION: The hES cell-derived antigen-presenting cells could be used to regulate alloreactive T cells and induce immune tolerance for improvement of the transplant acceptance of hES-cell derivatives.

Antigen-Presenting Cells↗

PubMedDate not supplied

Detection of dual-gene expression in arteries using an optical imaging method.

We evaluate the in vivo use of an optical imaging method to detect the vascular expression of green fluorescent protein (GFP) or red fluorescent protein (RFP), and to detect the simultaneous expression of GFP and RFP after transduction into arteries by a dual-promoter lentiviral vector driving their concurrent expression. This method involves using a charge-coupled device camera to detect fluorescence, a fiber optic probe to transmit light, and optical filters to distinguish each marker. In animal models, these vectors are locally delivered to target arteries, whereas the gene for a nonfluorescent cell-surface protein is transduced into contralateral arteries as the sham control. The images show distinct areas of bright fluorescence from GFP and RFP along the target arteries on excitation; no exogenous fluorescence is observed in the controls. Measured signal intensities from arteries transduced with the single- and dual-promoter vectors exceed the autofluorescence signal from the controls. Transgene expression of GFP and RFP in vivo is confirmed with confocal microscopy. We demonstrate the use of an optical imaging method to concurrently detect two distinct fluorescent proteins, potentially permitting the expression of multiple transgenes and their localization in the vasculature to be monitored.

Animals↗