ABSTRACT: 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.
To investigate the biological function of CD90(+) and CD90(-) keratinocytes.
CD90(+) and CD90(-) keratinocytes were purified from adult skin and cultured keratinocytes using fluorescent activated cell sorting, and their biological abilities were analysed using both in vitro and in vivo assays.
Flow cytometry (FCM) analysis identified approximately 18% of post-primary neonatal keratinocytes as CD90(+). However, during expansion of the culture, the expression level of CD90 rapidly decreased to about 2.5% at passage 10, while most of the keratinocytes maintained expression of alpha6 integrin. Purified CD90(+) keratinocytes demonstrated a sixfold higher cell growth rate than CD90(-) cells and the ability to form large (over 3 mm in diameter) colonies. We then quantitatively evaluated both populations using a previously described in vivo human epidermal cyst formation assay. Enhanced green fluorescent protein (EGFP)-labelled CD90(+) or CD90(-) keratinocytes were subcutaneously injected into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Six weeks after transplantation, EGFP(+) cell clusters in human epidermal cysts were evaluated using image analysis software. EGFP(+) cell cluster areas in the basal layer, derived from EGFP(+) CD90(+) cells, were eightfold larger than clusters of EGFP(+) CD90(-) cells. Furthermore, immunohistochemical staining and FCM analysis indicated that CD90 was expressed in most of the basal layer of the normal human epidermis.
These results indicated that CD90 is a useful marker for the detection of human KSC-enriched populations in cultured human keratinocytes.
British Journal of Dermatology 07/2006; 154(6):1062-70. · 3.67 Impact Factor
ABSTRACT: 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.
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.
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.
Gene-modified DCs derived from ex vivo expanded CB CD34(+) cells transduced by lentiviral vector will be useful in future immunotherapy protocols.
Experimental Hematology 11/2001; 29(10):1210-7. · 2.90 Impact Factor
ABSTRACT: 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 x 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 x 10(6) 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.
Cell Transplantation 02/2001; 10(4-5):409-12. · 5.13 Impact Factor