Precise analysis of human CD34-negative (CD34 ؊ ) hematopoietic stem cells (HSCs) has been hindered by the lack of a simple and reliable assay system of these rare cells. Here, we successfully identify human cord blood-derived CD34 ؊ severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with extensive lymphoid and myeloid repopulating ability using the intra-bone marrow injection (IBMI) technique. Lineage-negative (Lin ؊ ) CD34 ؊ cells did not show SRC activity by conventional tail-vein injection, possibly due to their low levels of homing receptor expression and poor SDF-1/CXCR4-mediated homing abilities, while they clearly showed a high SRC activity by IBMI. They generated CD34 ؉ progenies not only in the injected left tibia but also in other bones following migration. Moreover, they showed slower differentiating and reconstituting kinetics than CD34 ؉ cells in vivo. These in vivo-generated CD34 ؉ cells showed a distinct SRC activity after secondary transplantation, clearly indicating the long-term human cell repopulating capacity of our identified CD34 ؊ SRCs in nonobese diabetic (

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ABSTRACT. Cellular sites of [3H]galactose incorporated into F9 embryonal carcinoma cells were determined by electronmicroscopic autoradiography. Fifty-six per cent of the label was on plasma membrane.Embryonal carcinoma (EC) cells, the stem cells of teratocarcinoma, resemble the multipotential cells of early embryos (1). EC cells are known to produce a class of high-molecular-weight carbohydrates, which carry certain cell-surface markers of the parent cells (2, 3). It has yet to be established, however, what per cent of the carbohydrates in these cells are located on the plasma membrane. Although Muramatsu et al. (4) prepared a plasma membrane fraction of these cells and had good recovery of the carbohydrate label into the fraction, the degree of contamination of other organellas into the membrane preparation was not determined. We here report the use of electron microscopic autoradiography to determine the site(s) of these carbohydrates.F9 embryonal carcinoma cells were cultured in 2-cm Falcon dishes and labeled with [6-3H]galactose (1 Ci/mmole) for 24 h as described elsewhere (4). Labeled cells attached to the dish were washed 5 times with Dulbecco's phosphate buffered saline. The amount of [3H]galactose-label incorporated was 3-5 •~ 105 cpm per dish.When [3H]galactose was added to a cell layer that was washed immediately, the ratioactivity remaining in the layer was less than 3 •~ 104 cpm. That 85 % or more of the label incorporated into the cell retained its original form was confirmed by paper chromatography after acid hydrolysis which had been performed as described previously (4). Therefore, we used the galactose-labeled cell layer for the autoradiographical examination in situ.Cells on the culture dish were first fixed with 4 % glutaraldehyde in 0.2 M sodium cacodylate buffer, pH 7.2 at 4•Ž for 30 min. They then were postfixed in 2 % OsO4 in 0.2 M sodium cacodylate buffer, pH 7.2 at 4•Ž for 30 min. These fixed cells were washed with 0.2 M sodium cacodylate buffer, pH 7.2 then dehydrated with ethanol, the concentration of which in H2O was increased from 50 % to 60, 75, 80, 95 and 100 %. The sample was dehydrated at room temperature for 10 min; treatment with 100 % ethanol was repeated three times.The dehydrated sample was embedded in Epok 533 (Ookenshoji, Tokyo, Japan)

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