Hiroko Miyatake

Expression of CD90 on keratinocyte stem/progenitor cells

Background  The identification and purification of keratinocyte stem cells (KSCs) that are capable of self‐renewal and maintenance of differentiating cell populations could contribute both to our understanding of the biology of these cells, and to significant clinical applications, such as the culturing of keratinocytes for transplantation to severe burn wounds. Here, we report the detection of CD90+ cells in cultured normal human epidermal keratinocytes and adult skin.

Efficient lentiviral transduction of human cord blood CD34(+) cells followed by their expansion and differentiation into dendritic cells.

OBJECTIVE To support immune reconstitution after cord blood transplantation, immunotherapy using gene-modified dendritic cells (DCs), the most potent antigen-presenting cells, can be a powerful strategy for preventing infection and recurrence. To investigate the applicability of lentiviral vector-transduced DCs compared to retroviral vectors, we transduced umbilical cord blood (CB) CD34(+) cells, then expanded and differentiated them into DCs. MATERIALS AND METHODS We transduced CB CD34(+) cells by vesicular stomatitis virus G-protein pseudotyped self-inactivating lentiviral vector or retroviral vectors carrying the enhanced green fluorescent protein gene. The cells were expanded in the stroma-dependent culture system and transferred to the culture condition for developing DCs. The efficiency of transduction and expression of the transgene in severe combined immunodeficiency (SCID) mice-repopulating cells (SRCs) and DCs were compared between lentiviral vector and retroviral vectors. Induced DCs were cocultured with allogeneic or autologous T cells to test the ability to present antigens. RESULTS CB CD34(+) cells transduced by lentiviral vector and expanded ex vivo sustained stable transgene expression and multipotentiality by assessing SRCs assay and clonogenic assay of bone marrow cells from the transplanted mice. DCs derived from these cells expressed green fluorescent protein and surface markers CD1a, CD80, and HLA-DR and showed potent allo-stimulatory activity as well as nontransduced DCs did. On the other hand, we did not detect transgene expression in SRCs and DCs transduced by retroviral vectors. CONCLUSION Gene-modified DCs derived from ex vivo expanded CB CD34(+) cells transduced by lentiviral vector will be useful in future immunotherapy protocols.

A simple and efficient purification of transduced cells by using green fluorescent protein gene as a selection marker.

Background : Simple and efficient method for the selection of transduced cells would greatly facilitate the clinical utilization of retrovirus vectors. We developed a therapeutic bicistronic retrovirus vector for Gaucher disease, MFG‐GC‐GFP, which contains the human glucocerebrosidase (GC) gene and the green fluorescent protein (GFP) gene of the jellyfish Aequorea victoria as a vital selection marker, and investigated its applicability as gene therapy for Gaucher disease.

Acute lethal injury of lung and liver in mice transplanted with ex vivo-expanded CTLs.

Clinical application of cytotoxic T lymphocytes (CTL) induced in vitro is extensively used for the treatment of viral infection and malignant diseases. We produced anti H-2d CTL in vitro from C57BL/6 (B6) splenocytes presensitized with (B6 × DBA/2) F1 (BDF1) splenocytes to establish a model system of CTL therapy. The specificity and cytotoxic activity were high enough (E/T ratio 1:1 = 38.8%) to induce graft versus host reaction. Though the total number of B6 splenocytes decreased by 0.27 during the 4 days of culture, the number of CD8+ lymphocytes increased 1.3-fold. When more than 5 × 106 cells of H-2d -reactive CTL were transplanted into BDF1 mice, mice died within 2 days postinduction. This lethal effect was not seen in the mice induced with ConA-stimulated T cells. Histological examination of the lungs and liver revealed massive infiltration of neutrophils in alveoli and the necrosis of hepatocytes. Therefore, this protocol was shown to be effective to produce alloantigen-specific CTLs and applicable to in vitro manipulation such as retrovirus-mediated gene transfer.

Ex Vivo Expansion of Human Cord Blood Stem Cells and Genetic Manipulation

To introduce extrinsic genes into hematopoietic stem cells (HSC) by using retrovirus vectors, an efficient system for expanding HSC is required. We have established a novel culture system in which the murine stromal cell line HESS-5 dramatically supports the rapid expansion of cryopreserved cord blood primitive progenitor cells (CB-PPC) in synergy with TPO/FL. Within 5 days of serum-free culture in this system, a 50- to 100-fold increase in CD34+/CD38− cells was obtained; colony-forming units in culture (CFU-C) and mixed colonies (CFU-GEMM) were amplified by 10- to 30 fold and 10- to 20 fold, respectively. To further assess the long-term repopulating ability of those expanded cells, we performed a long-term culture-initiating cells (LTC-IC) assay and SCID-repopulating cells (SRC) assay using CD34+ cells cultured in this system. Within 5 days of culture, 5.1-fold amplification of the LTC-IC was obtained. SRC and their multilineage differentiation were detected in NOD/SCID mice 7 weeks after injection of these cultured cells. This system is further applicable to retrovirus mediated gene transfer to CB-PPC. The transduction efficiency of CD34+ cells was more than 40% when they were infected on HESS-5 monolayer cells. The engraftment of transduced cells in NOD/SCID mice 10 weeks after transplantation were confirmed by the presence of the gene introduced. These results indicate that this xenogeneic coculture system, in combination with human cytokines, can rapidly expand CB stem cells and is applicable to the efficient retrovirus-mediated gene transfer to them.